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STEM CELLS IN THE NEWS |
a Executive Director, Cell Therapy R & D Head, Hematopoiesis Department Holland Laboratory, American Red Cross hawleyr{at}usa.redcross.org
b Research Communications Manager, Holland Laboratory, American Red Cross sobieski{at}usa.redcross.org
SUCCESSFUL HEMATOPOIETIC STEM CELL GENE THERAPY FOR SEVERE COMBINED IMMUNODEFICIENCY
What a difference 3 years makes for the burgeoning field of gene therapy. Hopes for the development of effective genetic therapies have often been intertwined with the expectation that stem cells will offer the most effective target cells for such therapeutic interventions. By their nature, somatic stem cells—transduced with a therapeutic gene ex vivo and introduced back into a patient—are expected to establish in the recipient a renewable resource of mature cells producing the needed protein.
In the April 18 issue of The New England Journal of Medicine, Hacein-Bey-Abina et al. [1] reported sustained correction in four patients with X-linked severe combined immunodeficiency (SCID) by transplantation of CD34+ bone marrow cells genetically engineered ex vivo to express the gene encoding the common receptor subunit for a number of cytokines. Lack of the common gamma (
c) chain prevents the formation of receptors for interleukin (IL)-2, -4, -7, -9, -15, and -21. This deficiency leads to absence of T cells, which require the interaction of IL-7 and its receptor as a survival, proliferation and differentiation signal, and of natural killer (NK) cells, which similarly depend upon the interaction of IL-15 and its receptor for growth and development. The condition is usually fatal by 1 year of age due to severe infections.
The international team, led by clinician scientists at the Hôpital Necker Enfants Malades in Paris, harvested bone marrow from each of five male patients, aged 1 to 11 months, and isolated CD34+ hematopoietic stem/progenitor cells to a purity of 99% by two successive positive immunomagnetic selection procedures. The CD34+ cells were prestimulated for 24 hours in the presence of stem cell factor (SCF), Flt-3 ligand (FL), polyethylene glycol-conjugated megakaryocyte growth and differentiation factor (clinical grade thrombopoietin [TPO]) and IL-3. The cells were then transduced over a period of 3 days with an amphotropic Moloney murine leukemia virus-based MFG(B2) oncoretroviral vector containing the c gene in the presence of protamine sulfate and recombinant human fibronectin fragments. The patients, who had not undergone any preparative conditioning regimens, received 14 x 106 to 38 x 106 CD34+ cells per kilogram of body weight.
In this follow-up to a preliminary report published in 2000 [2], the researchers showed that the T-cell deficiency was corrected in four of the five patients analyzed over a period of up to 30 months following the infusion of the MFG(B2)c-transduced CD34+ cells. These patients showed clinical improvement: infections, lesions and related conditions cleared and did not recur. They were able to leave the sterile environment, and intravenous immunoglobulin G (IVIG) was discontinued 3 to 4 months after treatment. All four of these patients are now living at home under ordinary conditions, and none of them have had severe infections. The procedure was unsuccessful in the fifth patient who had a disseminated bacille Calmette-Guérin infection. He subsequently received an unrelated allotransplant which partially restored his T cell immunity.
The four patients who showed clinical improvement had a normal distribution and numbers of T cells, which exhibited functional responses similar to those of T cells in age-matched controls. Similarly, NK cells were detectable in all four patients. Virtually all of the T cells and NK cells expressed the c transgene, indicating that it conferred a selective growth advantage to the cells.
At the 5th Annual Meeting of the American Society of Gene Therapy (ASGT), held in Boston from June 5 to 9, the group reported the results of high resolution vector integration site analysis by linker adaptor-mediated polymerase chain reaction (LAM-PCR) methodology [3]. They detected multiple (20-40) LAM-PCR replicons in peripheral blood mononuclear cell and CD3+ T cell samples, indicating multiclonal reconstitution with transduced T-lineage cells. In one of the patients (patient 2), two unique integration sites identified in CD3+ T cell samples at 13, 24, and 32 months posttransplant were also present in individual myeloid colonies arising from 5-week long-term culture-initiating cells (LTC-ICs) derived 21 months posttransplant. These findings provide the first demonstration of successful gene transfer to human multilineage repopulating cells with therapeutic correction of disease. What is especially noteworthy about these results is that enhanced engraftment was observed and there were apparently fewer treatment-related complications compared to bone marrow transplantation with haploidentical samples, where reconstitution of T cells is delayed by 2 months or more, is never complete, and is frequently accompanied by graft-versus-host disease [4].
Parsley et al. reported at the ASGT meeting that they had also successfully treated X-linked SCID by oncoretroviral vector-mediated transduction of CD34+ bone marrow cells [5]. The transduction conditions were comparable to those used by Hacein-Bey-Abina et al. except that the vector particles were pseudotyped with the envelope protein from the gibbon ape leukemia virus, which may be more efficient than amphotropic vector particles for gene delivery to human hematopoietic stem cells [6]. The vector backbone also differed slightly from the one used by Hacein-Bey-Abina et al. in that it did not contain the modified primer binding site (B2 mutation, a G to A transition at position +160) present in the former, which had previously been shown to lead to significantly improved maintenance of transgene expression in murine bone marrow transplant recipients [7]. Both patients were clinically well with near normal immunological parameters at periods over 6 months post-infusion. Although multilineage reconstitution with transgene-corrected cells needs to be confirmed and longer follow-up times are required, these preliminary observations may indicate that downregulation of transgene expression due to methylation associated with the wild-type oncoretroviral primer binding site is a peculiarity of the murine system [8].
Also presented at the ASGT meeting were encouraging outcomes of two SCID patients with adenosine deaminase (ADA) deficiency who had been treated by CD34+ cell gene therapy. Aiuti et al. [9] transduced autologous CD34+ bone marrow cells with an oncoretroviral vector containing the ADA gene and the neomycin resistance gene using the SCF, FL, TPO and IL-3 cocktail combination and the 24-hour prestimulation/3-day transduction protocol on recombinant human fibronectin fragments. At days -3 and -2, the patients received nonmyeloablative conditioning with busulfan (4 mg/kg total dose). Patient 1 received 8.6 x 106 CD34+ cells per kilogram of body weight, and patient 2 received 0.9 x 106 CD34+ cells per kilogram of body weight. For patient 1, in which a 20 month follow-up was possible, peripheral blood lymphocytes (PBLs) returned to normal levels, accompanied by a normal distribution of CD4+ and CD8+ T cells, B cells, and NK cells. Vaccination with tetanus toxoid and assessment of proliferation responses to polyclonal antigens indicated that most immune system functions had normalized, allowing discontinuation of IVIG therapy. The frequency of transduced cells in patient 1 was almost 100% in T, B, and NK cells, and ranged from 5%-15% in the non-lymphoid lineages. At day 330, CD34+ bone marrow cells collected from this patient and transplanted into immunodeficient bone/thymus-SCID-hu mice were able to give rise to transduced mature B and T cells, indicating transduction of SCID-repopulating cells.
Importantly, polyethylene glycol (PEG)-bovine ADA replacement therapy was not available to these patients. Previous efforts to correct ADA-SCID by transduction of autologous PBLs have only been partially successful [10], and it has been presumed that this has been due in part to the absence of a selective advantage of the transduced cells because of PEG-ADA administration. In this context, in the May issue of Nature Medicine, Aiuti et al. had reported successful immune reconstitution in an ADA-SCID patient who had received autologous oncoretroviral vector-transduced PBLs that had reached a plateau of 1%-3% [11]. Although there was evidence of sustained expression of the ADA transgene, the patient continued to exhibit lymphopenia with depressed response to mitogens and showed no T-cell response to nominal antigens. The researchers therefore decided to discontinue the patient's PEG-ADA treatment in hopes of boosting immune function.
PEG-ADA dosages were decreased over time and, after 3 months, discontinued entirely. Following discontinuation of PEG-ADA treatment, the patient continued in good clinical condition and minor upper respiratory infections resolved normally with standard oral antibiotic therapy. Symptoms of asthma and perioral eczema continued to decrease. The investigators used fluorescence in situ hybridization and T-cell clone molecular analysis to assess the ratio of transduced to non-transduced T lymphocytes in the patient. They observed that transduced T cells increased with respect to non-transduced T cells while enzyme replacement was decreased, and eventually replaced non-transduced T cells altogether after enzyme therapy was discontinued. The levels of ADA produced by the cells were sufficient to fully restore T-cell function. Despite the successful remediation of T-cell function in this patient, a subsequent diagnosis revealed increased intracranial pressure associated with accumulation of red blood cell deoxyribonucleotides, a characteristic typical in ADA-SCID patients. PEG-ADA therapy was resumed for a time, during which selective advantage of the transduced T cells was lost. After enzyme therapy was discontinued a second time, the pool of transduced lymphocytes continued to expand again and red blood cell deoxyribonucleotides appeared to stabilize at a lower level than previously observed.
The combined results of these clinical gene therapy trials for ADA-SCID indicate that under conditions of efficient functional ADA gene transfer, discontinuation of enzyme therapy can contribute to correction of immune function. While providing proof-of-principle, the procedure for PBL gene therapy for ADA-SCID is such that patients typically receive 10 to 13 infusions of culture-expanded transduced T cells. With these new reports demonstrating immune reconstitution in ADA-SCID and successful treatment of X-SCID by a single infusion of short-term cultured CD34+ bone marrow cells, the field can breathe a collective sigh of relief: a milestone has been reached that heralds the beginning of the era of gene therapy.
THERAPEUTIC CLONING APPROACH PARTIALLY CORRECTS SCID IN MICE
In the April 5 issue of Cell, scientists at the Massachusetts Institute of Technology-affiliated Whitehead Institute for Biomedical Research reported efforts toward the correction of SCID in mice by nuclear transfer-facilitated combination cell and gene therapy [12]. Rideout et al. successfully treated Rag2-/-c-/- immunodeficient mice by transplantation of hematopoietic precursors derived from genetically modified Rag2-/- embryonic stem (ES) cells generated by somatic nuclear transfer technology.
Rag2-/- mice, which are nullizygous for the Rag2 recombinase gene, are devoid of B and T cells and have an immunodeficiency that resembles Omenn syndrome in humans [13]. The researchers began by transferring nuclei derived from tail-tip cells of Rag2-/- mice into enucleated mouse ooctyes, which were allowed to develop to the blastocyst stage on mouse embryonic fibroblast feeder layers. The efficiency of this process was approximately 13%. One of the cloned blastocysts produced an isogenic Rag2-/- nuclear transfer ES (ntES) cell line. These cells were then converted to Rag2+/– ntES cells by targeted homologous recombination followed by Cre recombinase-mediated elimination of the loxP-flanked selectable marker. The repaired ntES cells were differentiated in culture into hematopoietic precursors [14] that were then engrafted into irradiated recipient mice. To accomplish engraftment, the researchers used an approach that was described in an accompanying article in the same issue of Cell. In the companion paper, Kyba et al. reported that ectopic expression of the HoxB4 homeobox gene in differentiating ES cultures conferred upon the primitive hematopoietic precursors the ability to engraft in irradiated Rag2-/-c-/- mice [15]. The basis for this strategy came from earlier studies demonstrating that enforced expression of HoxB4 resulted in enhanced in vivo regenerative potential of bone marrow-derived (i.e., definitive) hematopoietic stem cells [16]. To accurately mimic the clinical situation, the engineered hematopoietic stem cells would normally have been infused into syngeneic Rag2-/- mice. However, at this point, the researchers encountered an obstacle. The problem appears to be related to the fact that ES cell-derived hematopoietic precursors express low levels of major histocompatibility complex class I antigens and are thus eliminated by host NK cells. The investigators attempted to decrease endogenous NK cell numbers in Rag2-/- animals by immunodepletion with an anti-NK cell antibody, but this produced engraftment levels of only 1.5%. This setback led to the subsequent use of Rag2-/-c-/- mice as recipients. As with human SCID patients who lack c, Rag2-/-c-/- mice are completely devoid of NK cells in addition to (B and) T cells, and therefore do not reject transplanted embryonic donor cells [17]. Interestingly, while essentially complete donor chimerism was achieved in the myeloid lineage with this experimental strategy, the frequency of donor lymphoid cells in the peripheral blood was much less, with T and B cell levels of only 0.09% and 2.4% observed, respectively. Nonetheless, some immune function was restored. Rearranged Ig heavy chain and T cell receptor chain loci were documented by PCR analysis, and IgM, IgG and IgA antibodies could be detected in the serum of the mice.
This combined cell and gene therapy approach toward the treatment of SCID is unquestionably a tour de force of technical virtuosity. Although the investigators were not successful at sufficiently immunodepleting NK cells in Rag2-/- mice to allow significant engraftment of the corrected hematopoietic precursors in a syngeneic host, one might envisage that an effective immunodepletion strategy could be developed. For this experimental strategy to have clinical applicability though, it will be essential to determine whether the nuclei of primitive ntES cell-derived hematopoietic precursors can be efficiently reprogrammed to become definitive hematopoietic stem cells with multipotential engrafting activity, either by exposure to an appropriate inductive microenvironment or by direct delivery of HoxB4 protein [18]. Homeobox gene transfer is not a clinical option since it carries a risk of leukemic transformation [19, 20].
Technical difficulties aside, the real question is whether such a complex therapeutic stratagem would ever have clinical relevancy. If the history of hematopoietic stem cell gene therapy is an accurate barometer, politics permitting, we should know the answer to this question in about 15 to 20 years [21].
DISCLAIMER
Any views and opinions expressed herein are those of the authors. They do not necessarily reflect the policies or position of the American Red Cross.
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REFERENCES
This article has been cited by other articles:
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  A. Ramezani, T. S. Hawley, and R. G. Hawley Performance- and safety-enhanced lentiviral vectors containing the human interferon-{beta} scaffold attachment region and the chicken {beta}-globin insulator Blood, June 15, 2003; 101(12): 4717 - 4724. [Abstract] [Full Text] [PDF]
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