Hematopoietic stem cell gene therapy: selecting only the best

Hematopoietic stem cell (HSC) gene therapy can potentially cure a variety of human hematopoietic diseases, such as sickle cell disease. Selection and expansion of gene-corrected HSCs has now been accomplished for the first time using HSC from large animals - dogs and humans - with a novel drug-resistance gene, MGMT, which is not expressed in normal HSCs (see the related articles beginning on pages 1561 and 1581). Highly efficient lentiviral transfer and expression of MGMT into relatively few HSCs led to repopulation of most of the hematopoietic compartment with gene-corrected cells following suitable drug treatment. This selection system may be useful in human clinical trials to permit gene therapy in autologous and allogeneic bone marrow transplantation settings.     

Correction of murine hemophilia A by hematopoietic stem cell gene therapy.

A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally delivered FVIII transgene offers the possibility of permanently correcting the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based FVIII oncoretroviral vector into immunocompetent hemophilia A mice that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy), or a nonmyeloablative preparative regimen involving busulfan. Therapeutic levels of FVIII (42, 18, and 11% of normal, respectively) were detected in the plasma of the transplant recipients for the duration of the study (over 6 months). Moreover, subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII inhibitor antibody response in any of the experimental animals, in contrast to the vigorous neutralizing humoral reaction to FVIII that was stimulated in naive hemophilia A mice. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.     

Hematopoietic stem cell therapy for malignant diseases

Allogeneic bone marrow or blood stem cell transplantation (SCT) has changed its face in the last two decades. The introduction of nonmyeloablative conditioning regimens has reduced procedure toxicity and allowed the application of SCT in patients and conditions in which SCT was not offered in the past. In this review we will summarize the changes and accomplishments achieved in the past years in the field of stem cell transplantation for malignant disorders.     

Hematopoietic stem cell therapy for malignant diseases

Allogeneic bone marrow or blood stem cell transplantation (SCT) has changed its face in the last two decades. The introduction of nonmyeloablative conditioning regimens has reduced procedure toxicity and allowed the application of SCT in patients and conditions in which SCT was not offered in the past. In this review we will summarize the changes and accomplishments achieved in the past years in the field of stem cell transplantation for malignant disorders.     

Hematopoietic stem cell

Hematopoietic stem cells (HSCs) maintain themselves over cell divisions (self-renewal) and produce all kinds of blood cells (multi-potency). Depletion of these cells eventually causes hematopoietic failure, while deregulated HSC division causes development of myeloproliferative disorders and leukemias. HSCs can be prospectively purified to nearly homogeneity in mice, but such a high-level purification has not been achieved in humans. HSCs are localized to an anatomical place called 'niche'. Specialized osteoblasts arrayed on the endosteum of cavernous bone and sinusoidal endothelial cells located at the distant position from the endosteum are the two representative candidates of such an HSC niche. A number of adhesion molecules and signaling molecules are thought to comprise the niche-HSC synapse. HSCs divide only once in 1-2 months. Both environmental signaling from the niche and HSC-autonomous molecular programs contribute to the quiescent state of HSCs, which is essential for the maintenance of HSC self-renewal capacity and homeostasis of blood production.     

Hematopoietic stem cell gene therapy in Hurler syndrome, globoid cell leukodystrophy, metachromatic leukodystrophy and X-adrenoleukodystrophy

Hurler syndrome, metachromatic leukodystrophy, globoid-cell leukodystrophy (Krabbe's disease) and X-linked adrenoleukodystrophy are inherited diseases of the CNS that can be cured or arrested by allogeneic hematopoietic stem-cell transplantation (HSCT). Despite significant progress in medical procedures and the availability of banked umbilical cord blood, HSCT is still associated with significant risks of graft failure or GVHD that can lead to death. Transplantation of autologous hematopoietic stem cells genetically modified to express the missing protein may circumvent the majority of the problems associated with allogeneic HSCT. Promising in concept, these strategies are now at a stage to be tested in phase I/II clinical trials to assess safety and potential efficacy.     

Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the TYMP gene, which encodes thymidine phosphorylase (TP). TP dysfunction results in systemic thymidine (dThd) and deoxyuridine (dUrd) overload, which selectively impair mitochondrial DNA replication. Allogeneic hematopoietic transplantation has been used to treat MNGIE patients; however, this approach has serious adverse effects, including the toxicity of myeloablative conditioning, graft rejection and graft-versus-host disease. With the aim of testing the feasibility of gene therapy for MNGIE, we transduced TP-deficient B-lymphoblastoid cells from two MNGIE patients, with lentiviral vectors carrying a functional copy of the human TYMP DNA coding sequence. This restored TP activity in the cells, which reduced the excretion of dThd and dUrd and their concentrations when added in excess. Additionally, lentiviral-mediated hematopoietic gene therapy was used in partially myeloablated double Tymp/Upp1 knockout mice. In spite of the relatively low levels of molecular chimerism achieved, high levels of TP activity were observed in the peripheral blood of the transplanted mice, with a concomitant reduction of nucleoside concentrations. Our results suggest that hematopoietic gene therapy could be an alternative treatment for this devastating disorder in the future.     

Hematopoietic stem cell expansion

This review addresses two issues: (1) cytokine-mediated expansion of functional end cells for the abrogation of short term neutropenia and thrombopenia following high dose chemotherapy and (2) cytokine-driven increase in absolute numbers of functional stem cells. The literature suggests that the short term exposure of CD34+ cells to cytokines produces mature progenitors which in turn give rise to functional neutrophils and platelets. The expansion of functional stem cells is a more complex issue, as primitive stem cells are quiescent and their growth requirements are less clearly defined.     

Hematopoietic stem cell transplantation in children

Medical and nursing care of the hematopoietic stem cell transplantation (HSCT) recipient are complex because of the pathophysiology, HSCT process, pre-HSCT conditioning regimens, numerous medications and therapies, acute and chronic complications, adverse effects, resources involved, and environmental considerations. The HSCT process and therapies may affect any body system, requiring proficient and prioritized nursing care, possibly in an intensive care setting. Understanding the timing of potential adverse effects and complications based on engraftment will help provide competent, high-acuity care. Although autogenic and allogeneic HSCT are curative treatment options, there are numerous morbidity and/or mortality risks throughout the HSCT journey.     

Micro-injection-mediated hematopoietic stem cell gene therapy

Over the past decade, significant attention has been devoted to the development of viral vectors (i.e., retrovirus, lentivirus, adeno-associated virus) and conditions capable of transducing hematopoietic stem cells. After several years of disappointing results, recent reports in humans and other primates, most particularly the French report of successful treatment of X-linked severe combined immune deficiency (SCID) [1.], indicate that viral approaches will be successful in treating specific hematopoietic diseases. However, it is clear that alternate non-viral methods of gene delivery and genetic modification offer significant advantages, and may in fact be the only effective approach for treating certain blood diseases. In this review, we focus on glass needle-mediated micro-injection as a method for the delivery of genetic material into blood stem cells, with an emphasis on molecules capable of either compensating gene deletions/mutations or directly repairing gene mutations.     

Hematopoietic stem cell transplantation.

Hematopoietic stem cells have been shown to provide the best chance for long term patient engraftment in bone marrow transplantation, peripheral blood stem cell transplantation, and umbilical cord blood transplantation. Characteristics of these cells include the ability to divide without differentiating; presence in bone marrow, peripheral blood, and cord blood; and negativity for all lineage CD markers. Autologous, syngeneic, and allogeneic transplantations have all been performed and patients respond well. Complications include graft rejection or failure and graft-versus-host disease; however, such complications can be minimized and treated. Hematopoietic stem cell transplantation holds promise for the treatment of many malignancies and metabolic diseases that have not responded to other forms of treatment.     

In vivo selection and chemoprotection after drug resistance gene therapy in a nonmyeloablative allogeneic transplantation setting in dogs

We have previously demonstrated successful in vivo selection, chemoprotection, and modulation of donor chimerism in dogs that received myeloablative allogeneic stem cell transplantation with cells expressing the P140K mutant of the DNA repair enzyme methylguanine methyltransferase (MGMTP140K). Here, we wished to investigate whether in vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after nonmyeloablative transplantation, which could allow for less toxic transplantation regimens for patients with malignant and genetic diseases. Three dogs received a nonmyeloablative conditioning regimen and infusion of allogeneic stem cells transduced with MGMTP140K. All three dogs had stable gene marking and donor chimerism before receiving a course of O(6) -benzylguanine (O(6) BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) between days 210 and 589 after transplantation. One to four doses led to a marked increase in gene marking in all dogs. Furthermore, the transduced cells conferred chemoprotection and prevented severe neutropenia. Our results suggest that drug resistance gene therapy is feasible and safe in the nonmyeloablative transplantation setting.     

Hematopoietic stem cell transplant in cancer treatment

Hematopoietic stem cell transplantation (HSCT) includes bone marrow, peripheral blood, cord blood, autologous, allogeneic, related or unrelated. These transplants have two main roles in cancer treatment, i.e., supportive treatment for marrow toxicity and immunotherapy for cancer cells. HSCT has also severe adverse reaction such as, bacterial fungal or viral infection by neutropenia, or graft versus host disease by the replacement of immune system. In autologous HSCT the main role is to prevent severe marrow toxicity which enables extreme high dose of anticancer drug administration. In allogeneic HSCT the main roles become both, support for marrow toxicity and immunotherapy, which possibly complicated with immunological adverse reactions. In mini-transplant or allogeneic transplant with reduced intensity, the main role is immunotherapy. These three kinds of transplants have each own indication and conducted in 17,472 patients till 2001 in Japan.     

Progress towards hematopoietic stem cell gene therapy

The introduction of recombinant genetic material into human cells for therapeutic purposes offers tremendous potential. However, almost from the beginning, the application of gene therapy has been characterized by the striking discrepancy between its promise and realization. Over the past 15 years, much has been learned about the various gene transfer systems and the requirements for efficient hematopoietic stem cell gene transfer. In the current review, we will summarize recent improvements in hematopoietic stem cell gene transfer, describe some of the promising results from recent clinical applications and the impediments that remain.     

In vivo selection to improve gene therapy of hematopoietic disorders

Successful gene therapy of hematopoietic disorders lacking intrinsic natural selection for genetically corrected cells will require efficient ex vivo gene transfer into autologous hematopoietic stem cells (HSCs). For these diseases, currently available gene transfer methodologies are unlikely to result in therapeutic numbers of corrected HSCs, especially in the setting of minimal recipient conditioning. A strategy to increase the numbers of genetically corrected HSCs in an individual is therefore highly desirable. One approach to overcome the barrier of limiting numbers of genetically corrected cells is to endow them with a competitive advantage conferred by inclusion of a 'selectable' gene in the therapeutic vector. Herein, we review recent progress in the development of in vivo selection systems, which hold promise in facilitating successful gene therapy.     

Hematopoietic stem cell processing and cryopreservation

Either bone marrow or peripheral blood may be harvested to provide hematopoietic stem cells (HSC) for autologous transplantation. Both, however, comprise heterogeneous cell populations. The HSC necessary for successful engraftment constitute a very small fraction of the cells harvested. After collection, the harvested cells usually undergo several processing steps to reduce the product volume, remove cells (such as mature blood cells or tumor cells), or to cryopreserve the cells for later reinfusion. Granulocytes and red blood cells, for example, survive cryopreservation poorly using freezing techniques designed for HSC. Therefore, bone marrows being cryopreserved must be depleted of mature blood cells to avoid toxicity from infusion of damaged mature blood cells. Mature blood cells may also impede the variety of tumor cell purging techniques currently being studied. These processings are designed to minimize the loss of HSC while achieving an appropriate HSC product for the individual patient. A number of apheresis devices and cell washers simplify the enrichment of HSC in the harvested cell products. In contrast, tumor cell purging techniques are not standardized between the various transplant centers. © 1992 Wiley-Liss, Inc.