Engraftment of gene-marked hematopoietic progenitors in myeloma patients after transplant of autologous long-term marrow cultures.

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.

Gene therapy for canine alpha-L-iduronidase deficiency: in utero adoptive transfer of genetically corrected hematopoietic progenitors results in engraftment but not amelioration of disease.

Canine alpha-L-iduronidase (iduronidase) deficiency is a model of the human lysosomal storage disorder mucopolysaccharidosis type I (MPS I). We used this canine model to evaluate the therapeutic potential of hematopoietic stem cell (HSC) gene therapy for enzyme deficiencies. In previous studies, iduronidase-deficient dogs infused with autologous marrow cells genetically modified to express iduronidase had long-term engraftment with provirally marked cells, but there was no evidence of proviral iduronidase expression or clinical improvement. The presence of humoral and cellular immune responses against iduronidase apparently abrogated the therapeutic potential of HSC gene therapy in these experiments. To evaluate HSC gene therapy for canine MPS I in the absence of a confounding immune response, we have now performed in utero adoptive transfer of iduronidase-transduced MPS I marrow cells into preimmune fetal pups. In three separate experiments, 17 midgestation fetal pups were injected with 0.5-1.5 x 10(7) normal or MPS I allogeneic long-term marrow culture (LTMC) cells transduced with neo(r)- or iduronidase-containing retroviral vectors. Nine normal and three MPS I pups survived the neonatal period and demonstrated engraftment of provirally marked progenitors at levels of up to 12% for up to 12 months. However, the proportion of provirally marked circulating leukocytes was approximately 1%. Neither iduronidase enzyme nor proviral-specific transcripts were detected in blood or marrow leukocytes of any MPS I dog. Humoral immune responses to iduronidase were not detected in neonates, even after "boosting" with autologous iduronidase-transduced LTMC cells. All MPS I dogs died at 8-11 months of age from complications of MPS I disease with no evidence of amelioration of MPS I disease. Our results suggest that iduronidase-transduced primitive hematopoietic progenitors can engraft in fetal recipients, contribute to hematopoiesis, and induce immunologic nonresponsiveness to iduronidase in MPS I dogs. However, the therapeutic potential of HSC gene transfer in this model of iduronidase deficiency appears to be limited by poor maintenance of proviral iduronidase gene expression and relatively low levels of genetically corrected circulating leukocytes.

Genetically corrected autologous stem cells engraft, but host immune responses limit their utility in canine alpha-L-iduronidase deficiency.

Canine alpha-L-iduronidase (alpha-ID) deficiency, a model of the human storage disorder mucopolysaccharidosis type I (MPS I), is an ideal system in which to evaluate the clinical benefit of genetically corrected hematopoietic stem cells. We performed adoptive transfer of genetically corrected autologous hematopoietic cells in dogs with alpha-ID deficiency. Large volume marrow collections were performed on five alpha-ID-deficient dogs. Marrow mononuclear cells in long-term marrow cultures (LTMCs) were exposed on three occasions during 3 weeks of culture to retroviral vectors bearing the normal canine alpha-ID cDNA. Transduced LTMC cells from deficient dogs expressed enzymatically active alpha-ID at 10 to 200 times the levels seen in normal dogs. An average of 32% of LTMC-derived clonogenic hematopoietic cells were provirus positive by polymerase chain reaction and about half of these expressed alpha-ID. Approximately 10(7) autologous gene-modified LTMC cells/kg were infused into nonmyeloablated recipients. Proviral DNA was detected in up to 10% of individual marrow-derived hematopoietic colonies and in 0.01% to 1% of blood and marrow leukocytes at up to 2 to 3 years postinfusion. Despite good evidence for engraftment of provirally marked cells, neither alpha-ID enzyme nor alpha-ID transcripts were detected in any dog. We evaluated immune responses against alpha-ID and transduced cells. Humoral responses to alpha-ID and serum components of the culture media (fetal bovine and horse sera and bovine serum albumin) were identified by enzyme-linked immunosorbent assay. Cellular immune responses to autologous alpha-ID but not neo(r) transduced cells were demonstrated by lymphocyte proliferation assays. To abrogate potential immune phenomena, four affected dogs received posttransplant cyclosporine A. Whereas immune responses were dampened in these dogs, alpha-ID activity remained undetectable. In none of the dogs engrafted with genetically corrected cells was there evidence for clinical improvement. Our data suggest that, whereas the alpha-ID cDNA may be transferred and maintained in approximately 5% of hematopoietic progenitors, the potential of this approach appears limited by the levels of provirally derived enzyme that are expressed in vivo and by the host's response to cultured and transduced hematopoietic cells expressing foreign proteins.

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses.

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.

Fractionation of Gene Modified Hematopoietic Autografts into Multiple Weekly Infusions Does Not Improve Engraftment in Unconditioned Canine Recipients.

Hematopoietic stem cell (HSC) gene therapy will require efficient transfer of genes to HSCs and long term engraftment and proliferation of genetically modified HSCs following adoptive transfer. We evaluated whether fractionation of grafts into 4-5 weekly infusions to non-myeloablated, autologous canine recipients would improve engraftment of genetically modified HSCs. Experimental animals and controls receiving a single infusion had similar levels of engraftment with ∼3-10% of marrow derived progenitors carrying transgene sequences for up to 29 months. There appears to be no improvement of engraftment of genetically modified HSCs in non-myeloablated large animal recipients by dose fractionation.

Retrovirus-mediated gene transfer into human hematopoietic stem cells.

Human hematopoietic stem cells genetically modified by retroviral-mediated gene transfer may offer new treatment options for patients with genetic disease. The potential of gene-modified hematopoietic stem cells as vehicles for gene delivery was first illustrated by the demonstration that hematopoietic systems of lethally irradiated mice can be reconstituted with retroviral vector transduced syngeneic bone marrow, and that these cells can in turn provide genetically marked progeny which persist in blood and marrow over extended time periods. In contrast, hematopoietic stem cells from large animals prove difficult to transduce with retroviral vectors and are consequently less likely to function as vehicles for long-term gene therapy. Indeed, clinically relevant levels of gene transfer into large animal and human hematopoietic stem cells has not been widely achieved. The need for improved retroviral vector systems and for understanding the biology of hematopoietic stem cell gene transfer continue to fuel intense research activity. Preliminary results from human stem cell gene marking and gene therapy trials currently underway are encouraging. This contribution reviews the underlying concepts relevant to retroviral-mediated gene transfer into hematopoietic stem cells. We survey the evolution of approaches for gene transfer into hematopoietic stem cells, from murine and large animal models to the first human clinical trials. Finally, we discuss new strategies which are currently being pursued.

In vitro maintenance and retroviral transduction of human myeloma cells in long-term marrow cultures.

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology, flow cytometry, and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR, G418 resistance, and X-gal staining was also within the expected range; 65%, 44% and 23%, respectively. Thus, there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients, expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase, and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient, individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer.

Preclinical assessment of human hematopoietic progenitor cell transduction in long-term marrow cultures.

Long-term marrow cultures (LTMCs) were established from 27 human marrows. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retroviral vectors were evaluated. LTMCs were assessed for viability, replication-competent retrovirus, progenitors capable of proliferating in immune-deficient mice, and gene transfer. The average number of adherent cells and committed granulocyte-macrophage progenitors (CFU-GM) recovered from LTMCs was 28% and 11% of the input totals, respectively. There was no evidence by marker rescue assay or polymerase chain reaction (PCR) of replication-competent virus production during LTMC. No toxicity to cellular proliferation due to the transduction procedure was observed. The adherent layers of LTMCs exposed to retroviral vectors were positive for proviral DNA by PCR and by Southern blot analysis. Fifty-three percent of 1,427 individual CFU-GM from transduced LTMC adherent layers were positive for vector-derived DNA. For neocontaining vectors, the average G418 resistance was 28% of 1,393 LTMC-derived CFU-GM. Forty percent of 187 tissues from 30 immune-deficient mice injected with human LTMC cells were positive for human DNA 4-5 weeks after adoptive transfer. These studies indicate that multiple exposures of human LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our results further support the use of transduced LTMC cells in clinical trials of hematopoietic stem cell gene transfer.