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Keywords:

  • gene therapy;
  • haematopoietic stem cells;
  • haemoglobinopathies;
  • immune deficiency syndromes;
  • lysosomal storage diseases;
  • retroviral vectors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography

Abstract. Kohn DB (Children’s Hospital and Keck School of Medicine, Los Angeles, CA, USA). Gene therapy for genetic haematological disorders and immunodeficiencies. J Intern Med 2001; 249: 379–390.

Gene transfer and autologous transplantation of haematopoietic stem cells (HSCs) from patients with genetic haematological disorders and immunodeficiencies could provide the same benefits as allogeneic HSC transplantation, without the attendant immunological complications. Inefficient gene delivery to human HSCs has imposed the major limitation to successful application of gene therapy. A recently reported clinical trial of gene transfer into HSCs of infants with X-linked severe combined immunodeficiency (SCID) has achieved immune restoration because of the selective outgrowth of the gene-corrected lymphocytes. Newer methods for manipulating HSCs may lead to efficacy for other disorders. The problems and progress in this area are reviewed herein.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography

Gene therapy is an emerging medical modality in which genetic diseases will be corrected by transfer of a normal version of the relevant gene into a patient’s somatic cells. Gene transfer into the haematopoietic stem cells (HSCs) of patients with congenital immune deficiencies, lysosomal storage diseases or haemoglobinopathies, followed by their autologous transplantation, could provide the same benefits as allogeneic transplantation, without the immunological complications of graft rejection, graft-versus-host disease, and post-transplant immunosuppressive therapy. Additionally, the ability to modify HSCs genetically would allow augmentation of the properties of the HSCs or their derivatives, such as increasing resistance to the myelosuppressive effects of chemotherapy or decreasing susceptibility to infection by pathogens, such as HIV-1.

Successful gene therapy with HSCs requires efficient gene delivery to a high percentage of long-term reconstituting HSCs, stable persistence of the gene as the HSCs undergo extensive proliferation, and an appropriate level of expression of the gene in the relevant cell types. Whilst retroviral vectors based upon the Moloney murine leukaemia virus (MLV) are capable of efficient and stable gene delivery to murine HSCs, they have failed to achieve the necessary gene transfer efficiency in human HSCs. This poor gene delivery to HSCs has imposed the major limitation to application of gene therapy using HSCs, although newer methods for manipulating HSCs during gene transfer may lead to better efficacy. Other gene transfer modalities, such as lentiviral vectors or direct microinjection into HSCs of either self-integrating or autonomously replicating plasmids or molecules capable of mediating gene correction may also lead to improved efficacy in the future.

Because of the limited capability for gene transfer to human HSC, the clinical diseases which are currently being studied (e.g. severe combined immunodeficiency, SCID) have been chosen with the foremost consideration that they have potential for deriving some therapeutic effect from gene transfer into a small percentage of HSCs. As the gene transfer tools improve, more prevalent disorders, such as Gaucher’s disease and sickle cell disease will be approachable.

Progress in gene therapy using HSCs (Fig. 1)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography
image

Figure 1.  Transduction of haematopoietic stem cells (HSCs) by retroviral and lentiviral vectors. Quiescent HSCs can be activated and induced to replicate with positively acting factors, such as the recombinant haematopoietic growth factors flt-3 ligand (F3L), c-kit ligand (KL) and thrombopoietin (TPO). Suppression of inhibitory factors, such as transforming growth factor beta (TGF-β), or prevention of intracellular accumulation of the cell cycle inhibitory protein p27kip can allow proliferation. Other cytokines (IL-3, IL7, GM-CSF, G-CSF) support lineage commitment and differentiation. Retroviral vectors can transduce HSCs only after activation and replication have been induced. Lentiviral vectors may be able to transduce quiescent HSCs or HSCs that have been activated but are not replicating. Stable gene transfer into HSCs will allow long-term (years) production of gene-transduced cells, whereas gene transfer into committed progenitor cells will only lead to short-term (days to months) production of gene-transduced cells.

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Improved conditions for the ex vivo transduction of human HSCs by retroviral vectors

For more than 10 years, it has been possible to transduce the majority of stem cells in murine gene transfer/BMT models, using retroviral vectors of sufficient titres (e.g. 106 infectious units mL–1), stimulation with the appropriate recombinant haematopoietic growth factors [e.g. interleukin-3 (IL-3) and interleukin-6 (IL-6)] and co-cultivation of the marrow cells directly upon monolayers of the vector-producing fibroblasts [1]. In contrast, work in large animal models (canine and rhesus) had achieved only 0.1–1.0% stem cell transduction until recently [2] (see also article by C. Dunbar, this volume). Clinical trials of gene marking of autologous haematopoietic cells have also shown extremely limited abilities to transduce long-lived human stem cells, with marking of 0.1–1% of cells seen [3]. The reasons for the greater difficulty in performing gene transfer into stem cells from the large animals and human subjects are unknown; possible explanations include lower numbers of receptors for the amphotropic virus, intracellular blocks to virus integration, or a lower percentage of stem cells in active cell cycle which can be transduced by retroviral vectors.

Methods to maximize gene transfer into human HSCs have been continually studied by investigators at many centres. Newly cloned cytokines, such as flt-3 ligand and thrombopoietin, have been shown to induce replication of primitive human HSCs far more effectively than the cytokines available and used in previous studies (IL-3/IL-6/c-kit ligand) which probably act on more mature, less long-lived progenitor cells [4–6]. Hanenberg et al. [7] have shown that the presence of a support matrix of recombinant fibronectin used to coat the cell culture dishes also increases the efficiency of gene transfer into human HSCs. Fibronectin binds to integrins on the surface of the stem cells via an RGD domain and to the negatively charged envelope glycoprotein on the retrovirus vectors via a heparin-like domain (Fig. 2). The co-localization of cell and vector increases gene transfer, presumably by increasing the collision rate.

image

Figure 2.  Retroviral-mediated gene transduction of haematopoietic stem cells (HSCs). HSCs bound to recombinant fibronectin fragments (jagged symbol) interact with retroviral vector particles in the presence of recombinant haematopoietic growth factors flt-3 ligand (F3L), c-kit ligand (KL) and thrombopoietin (TPO).

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Additionally, these newer cytokines and the fibronectin support matrix each act to support HSCs survival during the ex vivo culture, resulting in a significantly greater level of stem cell engraftment after infusion of the cells [8]. The better survival of the stem cells during culture also allowed extension of the time of culture from the 3-day period used previously to a 5-day culture, during which time more of the primitive stem cells may begin to divide and become susceptible to retroviral-mediated transduction [6]. The net result of greater gene transfer efficiency and better survival of the HSCs should be a significant increase in the level of gene-containing stem cells engrafted in the subjects. These techniques have resulted in 10- to 50-fold higher frequencies of gene-containing cells in primate gene transfer/BMT studies [910]. In addition, the newer culture conditions are based on a serum-free approach, eliminating fetal calf serum, so that bovine antigens are not introduced to subjects with the infused cells, and thus immunological consequences are obviated.

Manipulation of cell cycle to increase retroviral-mediated gene transduction of human HSCs

Jan Nolta and co-workers have developed a novel method to increase gene transfer into primitive quiescent human HSCs [11]. The major effort to date had been to add stimulatory haematopoietic growth factors to induce cell division, which is required for murine retroviral vector to gain access to a cell’s chromosomes. The alternative approach explored by these investigators involved blocking the action of inhibitory factors that maintain the cellular quiescence. Prior studies had shown that TGF-β, produced both by the HSCs (autocrine) and by other cells in bone marrow (paracrine), suppress HSC proliferation and the inhibitory effects of TGF-β could be partially overcome with neutralizing antibodies [12]. Dao et al. [11] showed that expression of p27kip by HSCs also inhibited their proliferation. Primitive HSCs (either CD34+ cells which survived the cell cycle toxic agent 4-hydroperoxycyclophosphamide or CD34+/CD38-cells) could be transduced by murine retroviral vectors within a 24-h period if both TGF-β and p27kip were blocked, using a rabbit polyclonal antiserum to TGF-β and phosphorothioate antisense oligonucleotides to p27kip. Importantly, the HSCs manipulated to cycle by this approach did not lose their stem cell capacity, based upon their ability to engraft and proliferate in an immune-deficient murine xenograft host.

Use of lentivirus vectors for gene transduction of HSCs

Data from numerous laboratories have demonstrated that HIV-1-based lentiviral vectors are superior to murine retroviral-based vectors for gene transfer to primitive quiescent human haematopoietic progenitor cells [13–15]. Various features of lentiviruses, such as nuclear localization signals in multiple virion proteins and the presence of a central polypurine tract generating a triple-stranded ‘DNA flap’ in the reverse transcribed genome, allow the genome of lentiviruses to enter the nucleus of nondividing cells [16,17]. Ideally, this will result in a higher level of gene transduction of pluripotent HSCs, although definitive proof for this has not been obtained, with studies to date using extended long-term culture or NOD/SCID xenografts measuring progenitors which may be more short-lived and easier to transduce that long-lived pluripotent HSCs.

Definitive evidence of the improved efficacy for gene transfer to human HSCs of lentiviral vectors will probably only be obtained in clinical trials. The major barrier to these studies is the concerns over the biohazards from using a vector derived from a pathogenic virus (HIV-1). The current ‘third-generation vectors’ have less than 5% of the HIV-1 genome in the delivery vector and less than 25% of the entire HIV-1 genome in the packaging construct (expressing gag and pol sequences only). All laboratory experience to date has found a complete absence of replication-competent lentivirus in more than 100 vector preparations. It is likely that lentiviral vectors will be evaluated in clinical trials in a few years, either for subjects with HIV-1 infection to introduce anti-HIV-1 genes or for subjects with genetic diseases where the level of gene transfer needed for a therapeutic benefit exceeds what may be achieved with murine retroviral vectors (e.g. Gaucher’s disease, haemoglobinopathies).

More effective expression from retroviral vectors transferred into HSCs

Retroviral vectors have two basic designs for expression: transcription from the retroviral 5′ LTR or from a secondary, ‘internal’ promoter derived from other viruses (e.g. CMV, SV40) or from cellular genes (e.g. PGK, β-actin, β-globin). In general, simple designs of retroviral vectors in which the 5′ LTR drives expression as the sole transcriptional unit (5′LTR-1-cDNA-3′LTR) can be produced to higher titres and are more likely to retain the integrity of their genomes than are more complicated vectors with internal promoters [18,19]. A drawback of LTR-driven vectors is that expression is constitutive, lineage nonspecific, not physiologically regulated and may be subject to silencing. Internal promoters could produce expression which recapitulates that of endogenous genes for lineage specificity, responsiveness to physiological stimuli and persistence.

Initial studies of gene transfer focused on ‘housekeeping enzymes’ such as adenosine deaminase (ADA) and glucocerebrosidase, which are ubiquitously expressed in a loosely regulated manner. Expression of these genes under control of constitutive viral promoters, such as the MLV LTR or the CMV or SV40 enhancer/promoters, may result in expression in all transduced cells at variable levels. For example, the MLV LTR drives the human ADA and GC cDNA to levels one to three times those of endogenous patient cells [20,21] and the human α-iduronidase cDNA to >500 times that of normal [22]. Such nonregulated expression may be tolerated for these enzymes. However, some other gene products may be harmful if expressed without lineage specificity or physiological control, e.g. haemoglobin or signal transduction pathway components, such as btk involved in X-linked agammaglobulinaemia. Additionally, as our group and others have observed, the MLV LTR may express poorly in primary haematolymphoid cells and is subject to ‘silencing’ over time after HSC transduction [23–25]. Therefore, it is important to develop retroviral vectors which can have suitable levels, longevity and lineage-restricted expression.

Our group and others have developed modified retroviral vectors with significantly improved expression activity, compared with the standard MoMuLV vector, in HSCs and their mature blood cell progeny, including T lymphocytes [26–28]. These vectors (such as MSCV and MND) contain the enhancers from the myeloproliferative sarcoma virus, which expresses more actively in haematopoietic and lymphoid cells than does the MLV LTR and has deletions of cis-inhibitory sequences such as the primer binding site. More effective expression could provide a more enduring effect, leading to greater selective advantage to gene-corrected T lymphocytes in SCID patients, higher levels of lysosomal enzymes for cross-correction of connective tissue, etc. Use of transcriptional control elements, such as lineage-specific promoters, locus control regions (LCRs), scaffold/matrix attachment regions (S/MARs) and insulators may lead to physiologically regulated gene expression (Fig. 3), which is likely to be required for correction of some disorders where indiscriminate, ubiquitous gene expression could be harmful (e.g. β-globin, btk, WASP) [29–31].

image

Figure 3.  Gene expression by an integrated retroviral vector provirus. Genetic elements such as locus control regions (LCRs), scaffold/matrix association regions (S/MARs) and insulators may shield gene expression by retroviral vectors from inhibitory effects of the flanking chromosomes.

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Direct microinjection of genes into HSCs

Whilst microinjection of DNA directly into the nuclei of target cells is used routinely in the generation of transgenic mice, it is done on a cell-by-cell basis, greatly limiting the number of cells which can be modified. Davis et al. [32] have recently shown that microinjection through glass needles can be used to introduce genes into human CD34+ haematopoietic progenitor cells, using a fibronectin matrix to reversibly immobilize the cells to allow their manipulation. Scale-up of this method by the use of robotic techniques coupled to high levels of enrichment for HSCs using immunophenotypic isolation (e.g. CD34+/CD38–/Thylow cells) may make microinjection of a sufficient number of HSCs for transplantation a realistic possibility. Microinjection could allow the elimination of viral vectors and permit the use of gene-correcting molecules, such as chimeric DNA/RNA which can repair mutations through homologous recombination [33]. Gene correction would leave the repaired endogenous gene within its normal chromosomal context where it should retain physiological expression patterns; in contrast, gene addition with viral vectors leads to ectopic gene insertion with susceptibility to positional effects on gene expression.

Clinical trials of gene transfer into HSCs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography

Studies in murine models of human haematopoietic and immunological diseases (e.g. XSCID, Jak3-deficient SCID, β-thalassaemia, MPS VII, CGD, Fanconi’s anaemia, erythropoietic protoporphyria) have shown partial or complete correction by retroviral-mediated gene transfer to HSCs [34–41]. However, the dichotomy between the successful gene transfer into mice and the poor gene transfer into human cells necessitates that potential benefits of gene therapy be evaluated in clinical trials with human subjects. Since the first trial involving gene transfer into human HSCs of subjects with ADA-deficient SCID in 1992 [42], there has been an ongoing iterative process between laboratory development of improved gene transfer and expression techniques and their evaluation in clinical trials (Table 1).

Table 1.   Candidate genetic disorders for gene therapy using haematopoietic stem cells Thumbnail image of

Gene therapy for SCID

Severe combined immunodeficiency refers to a group of monogenic inherited disorders with absent T- and B-lymphocyte function and extreme susceptibility to opportunistic infections. From more than three decades of experience with the application of allogeneic bone marrow transplantation for SCID, it has been known that T lymphocytes derived from genetically normal HSCs have a profound growth advantage over the genetically deficient cells and that a small number of normal cells may lead to re-population and reconstitution of the immune system (Fig. 4) [43]. SCID has been an initial candidate disease for early clinical trials of gene transfer into autologous HSCs, because the expected selective survival advantage for gene-corrected T lymphoid progenitor cells may allow clinical benefit to be achieved despite low-frequency transduction of HSCs.

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Figure 4.  Gene transfer may allow selective expansion of progeny cells from a few transduced haematopoietic stem cells (HSCs). Selective expansion from HSCs by introduction of a gene which corrects a physiological proliferative or survival function (e.g. severe combined immunodeficiency, SCID) confers resistance to a selective agent (e.g. chemotherapy) or induces increased proliferation (e.g. growth factor receptor). Cells lacking the gene fail to survive, are inhibited or are outgrown.

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Adenosine deaminase (ADA)-deficient SCID

The first aetiological form of SCID to be addressed was that caused by deficiency of ADA, because it was the only form for which the relevant gene had been cloned at that time and precise gene expression regulation was not thought to be required for safety and efficacy. The first clinical trial of gene therapy, begun in 1990, involved two young girls with this disorder, using their peripheral blood T lymphocytes as the target for ADA gene transfer [44]. Multiple courses of leucopheresis, transduction and re-infusion of the cells led to increased numbers and functional activities of T lymphocytes. In at least one of the patients, ADA-transduced T lymphocytes have remained at a relatively stable level of 10–20% of total T lymphocytes, now almost 10 years since her treatment first began. However, these patients remain on full dosages of PEG-ADA enzyme replacement, making it difficult to attribute immunological functions to the gene transfer.

Despite these encouraging results using mature T lymphocytes, genetic correction of HSCs presents the potential for creating a lifelong source of ADA expressing T lymphoid precursors, which may produce a broad immunological repertoire. In the early 1990s, five ADA-deficient SCID patients were treated by insertion of the ADA gene into their bone marrow cells (two in Italy and three in The Netherlands), and one of the original patients who received ADA gene therapy via T lymphocytes has been treated by insertion of a different ADA vector into her G-CSF-mobilized peripheral blood stem cells [42,45]. The two patients treated in Italy have shown continued production of T lymphocytes containing the introduced ADA cDNA, with gradual withdrawal of PEG-ADA underway [42]. Only low extents of gene transfer were achieved in the three patients treated in The Netherlands, with minimal evidence of in vivo production of transduced cells for less than 6 months [45]. No adverse reactions have been noted to date in these ADA-deficient patients or any of the other patients who have received cells exposed to retroviral vectors.

In 1993, our group at Children’s Hospital Los Angeles, working with Michael Blaese and coworkers at the National Institutes of Health, USA, performed a clinical trial of gene transfer of the normal human ADA cDNA into CD34+ cells from the umbilical cord blood of three ADA-deficient newborns [46]. The methods used for ex vivo transduction of the CD34+ cells had been developed in our laboratory and involved culturing the cells with a mixture of recombinant cytokines (IL-3/IL-6/c-kit ligand), which induced proliferation of the HSCs to allow retroviral transduction. We found that all three infants engrafted with gene-corrected HSCs, based on the production of haematopoietic and lymphoid cells of multiple lineages through the present time. The frequency of gene-containing leucocytes in the peripheral circulation has been quite low, in the range of 1/10 000 for cells of the myeloid lineages which do not require ADA. In contrast, the frequency of T lymphocytes containing the normal ADA gene was significantly higher, in the range of 1/100–1/10, clearly demonstrating the selective survival advantage conferred to the gene-corrected T lymphocytes [47]. When we withdrew PEG-ADA from one subject for a 2-month period, the number of T lymphocytes and their responses to PHA were preserved, although the numbers of natural killer (NK) cells and B lymphocytes decreased. RT-PCR analysis of PBMC revealed that the vector was not being expressed, although expression was strongly induced by in vitro activation of T lymphocytes with phytohaemagglutinin (PHA) and IL-2 [47]. We conclude that the gene transfer methods and expression by the vector used in the first trial were insufficient to produce clinical benefit, although safety and feasibility were shown (Fig. 5).

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Figure 5.  Haematopoiesis and gene transduction. Haematopoietic stem cells (HSCs) undergo self-renewal or commitment to differentiation. Progenitor cells proliferate and differentiate along pathways of increasingly restricted developmental potential to produce mature blood cells.

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Since the time of those trials, newer methods have been developed for more effective retroviral-mediated gene transfer and expression (described above), and may allow better efficacy for ADA gene transfer. Our group at Children’s Hospital Los Angeles, in collaboration with Fabio Candotti and coworkers at the National Human Genome Research Institute, NIH, USA, is starting a second-generation trial for ADA-deficient SCID using these improved methods for gene transfer and expression in HSCs.

X-linked SCID (XSCID)

Approximately 40% of cases of SCID are inherited in an X-linked pattern [48]. The gene responsible for the X-linked form of SCID was identified in 1993 as a third chain of the multimeric receptor for IL-2 (initially named the IL-2 receptor γ chain) and was subsequently shown to be a common component of the receptors for cytokines IL-2, IL-4, IL-7, IL-9 and IL-15 (and therefore termed the common cytokine receptor gamma chain or γc) [49]. Preclinical studies showed that retroviral-mediated transfer of a normal human γc cDNA corrected functional defects in T lymphocytes and NK cells from XSCID patients and in T cells produced from transduced HSCs in γc gene knock-out mice [50–52].

A recent trial of gene transfer into bone marrow from children with XSCID, using essentially the same techniques which have resulted in improved transduction in primate models (cytokines including c-kit ligand, Flt-3 ligand and thrombopoietin on recombinant fibronectin), has led to early reconstitution of the immune system with gene-containing T lymphocytes [53]. After 10 months of follow-up, both subjects had recovered essentially normal T-cell function, with the γc transgene detected in circulating T and NK cells. The tempo of immunological reconstitution following infusion of the genetically corrected autologous HSCs was quite similar to that seen in SCID patients following transplantation of allogeneic bone marrow from HLA-matched siblings.

At the third annual meeting of the American Society of Gene Therapy in June 2000, lead investigator Alain Fischer updated the published results to report on a total of five treated XSCID subjects. The immune restoration reported in the first two subjects has persisted and has been replicated in two additional subjects. A fifth subject showed only low-level production of gene-corrected cells; the lack of success in this subject was attributed to loss of the infused transduced HSCs due to significant hepatosplenomegaly from an ongoing BCG infection.

If these results are sustained, they would represent a major milestone in the field of gene therapy as the first clinically significant benefit for a genetic disease by gene transduction of HSCs. It is possible that gene transfer occurred into lymphoid lineage-restricted progenitor cells of finite life span, rather than pluripotent HSCs of long-lived potential. Transduction only of progenitors would be expected to limit the duration of generation of new T lymphocytes and could eventually lead to ‘holes’ in the immunological repertoire. Thus, extended follow-up will be needed to determine whether these patients are truly cured. However, the findings so far are quite exciting and signal that the promise of gene therapy as a new treatment for genetic diseases may, at last, be fulfilled.

The logic of targeting SCID as an early candidate disease for gene therapy using haematopoietic stem cells has been substantiated by these highly promising results in infants with XSCID. Although ADA-deficient SCID has been under study for a longer time than XSCID (in part due to the earlier cloning of the responsible ADA gene), it remains unclear whether a similar clinically beneficial result will be seen in our upcoming ADA gene transfer trial. XSCID may afford a stronger intrinsic selective advantage for corrected cells than ADA deficiency. PEG-ADA enzyme therapy will blunt the selective advantage of corrected cells whilst it is being given. Thus, a higher level of gene transfer may be needed to correct ADA deficiency.

Chronic granulomatous disease

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography

Chronic granulomatous disease (CGD) is due to defects in neutrophil antimicrobial oxidase activity, leading to multiple chronic purulent infections [54]. The genes responsible for the X-linked and three autosomal forms of CGD have been cloned and gene knock-out mice models developed. Whilst palliative therapy with antibiotics and surgical drainage of abscesses, coupled with the use of gamma-interferon which partially restores antimicrobial activity, may be beneficial, only allogeneic HSC transplantation has been curative. Studies of retroviral-mediated correction of HSCs in murine models of the X-linked and gp91phox-deficient forms of CGD have shown significant phenotypic improvement, including increased resistance to infections [38,39,55]. A series of clinical trials have been performed by Malech et al. [56] in the National Institute of Allergy and Infectious Diseases at the NIH, USA, using retroviral-mediated delivery of the normal p47phox cDNA to patient CD34+ PBSC. Clear-cut documentation has been obtained of the production of gene-corrected neutrophils expressing normal oxidase activity in subjects, although at low levels and for only a few months. There have been some suggestions of clinical improvements in ongoing infections, suggesting that gene transfer into short-lived myeloid progenitor cells, which may be more readily transduced than long-lived pluripotent HSCs, may serve as a ‘bridging’ therapy during acute infections.

Gaucher’s disease

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography

Gaucher’s disease is the most common lysosomal storage disease [57]. Genetic deficiency of the lysosomal enzyme glucocerebrosidase results in storage of its substrate glycolipid in resident tissue macrophages of the spleen, liver and bone. Allogeneic BMT has been shown to be capable of significant benefit in Gaucher’s disease patients, with turnover of the lipid-laden host macrophages by replacement with normal donor-derived macrophages derived from the transplanted bone marrow [58,59]. The risks of allogeneic BMT, including graft rejection or graft-versus-host disease, have limited the use of transplants mainly to patients with the more severe ‘slow onset’ neuroneopathic subtype of Gaucher’s disease. Because gene therapy with autologous HSCs would not engender immunological reactions, it may be safer than allogeneic BMT. Whilst there is no theoretical basis to predict a selective survival advantage to gene-corrected cells in this disorder, the presence of small numbers of genetically normal cells could result in increased substrate breakdown and clinical benefits.

Three groups performed clinical trials of retroviral-mediated transfer of the normal glucocerebrosidase cDNA into CD34+ cells from patients with Gaucher’s disease [60,61]. Cells were transduced by ex vivo culture in medium containing cytokines IL-3, IL-6 and c-kit ligand, and no cytoreductive conditioning was given prior to cell re-infusion. Gene-containing cells were detected in peripheral blood only transiently and at very low levels. The disappointing findings from these studies suggest that future approaches to Gaucher’s disease may need to be withheld until significant improvements in gene transfer efficiency to HSCs are assured.

Fanconi’s anaemia

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography

Fanconi’s anaemia (FA) comprises a group of disorders due to inherited defects in genes involved in the DNA repair response which have marrow failure and progression to leukaemia as key features [62]. Allogeneic bone marrow transplantation from fully matched sibling donors can be curative for the majority of patients [63]. Normal haematopoiesis is restored from the donor marrow and the heightened risks of leukaemia are reversed, although subjects remain at increased risk for other malignancies. In contrast, the results to date have been significantly poorer for FA patients lacking an HLA-matched family donor, who therefore undergo HSC transplants using nonfamily donors, mainly because of increased morbidity from graft-versus-host disease.

Because of the defective haematopoiesis of the FA HSCs, gene-corrected cells may have a selective survival advantage, leading to a relative increase in their contribution to haematopoietic cell production. Genes for most of the common genetic subtypes of Fanconi’s anaemia have been cloned and murine gene knock-out models developed. Retroviral-mediated insertion of FAA cDNA had led to phenotypic improvement in haematopoietic cells from FA patients and in gene knock-out mice [40,64]. Competitive marrow repopulation studies in mice using mixtures of normal and FA gene knock-out marrow showed that there was only minimal selective growth advantage for the normal marrow after transplant [65]. However, subsequent imposition of haematopoietic stress of the marrow, by either serial transplantation or administration of the DNA clastogenic agent mitomycin C, revealed a significant growth advantage of the genetically normal marrow. Thus, as with SCID, FA patients may benefit from relatively low gene transfer, if pancytopenia were to be improved by growth from a small number of corrected cells.

One clinical trial has performed retroviral-mediated gene transfer in four subjects with FA group C [66]. CD34+ G-CSF mobilized PBSC were transduced and re-infused in three to four cycles without cytoablation. Gene-containing cells were present only transiently in peripheral blood and bone marrow. Interestingly, one patient received radiation therapy for a concurrent malignancy and had a transient appearance of gene-containing cells in blood and marrow, suggesting relative radiation resistance was conferred to haematopoietic progenitor cells by the gene transfer.

One unanswered question concerning the use of gene-corrected autologous HSCs into nonablated FA subjects is the unknown effects on the intrinsic leukaemogenic potential of the endogenous marrow. In allogeneic BMT, the cytoablative conditioning given to allow engraftment eradicates the endogenous marrow. It remains to be proven whether the presence of genetically corrected HSCs producing sufficient blood cells to eliminate pancytopenia would alleviate haematopoietic stress on the endogenous marrow and reduce its leukaemic potential. This unknown risk of development of leukaemia would need to be weighed against the benefits afforded by the avoidance of chemotherapy or radiation.

Haemoglobinopathies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography

The haemoglobinopathies, sickle cell disease and thalassaemia, constitute the most prevalent of the genetic diseases which may be treated with gene therapy using HSCs.

However, they are also amongst the most technically challenging, as fairly high levels of gene-corrected cells may be needed (e.g. 10–30%) and the transferred globin gene may need to be expressed only in erythroid cells at relatively precise levels to match that of the other globin chain. Only modest success has been achieved with efforts to develop retroviral vectors carrying the necessary transcriptional control sequences from the globin locus found to be required for appropriate expression in transgenic mice [67,68]. Recently, May et al. [69] reported that lentiviral vectors are capable of carrying these sequences, leading to improvement of anaemia in β-thalassaemic mice. Thus, lentiviral vectors may afford both the efficient gene transfer and the regulated gene expression required for effectively treating haemoglobinopathies by transduction of autologous HSCs.

Bibliography

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in gene therapy using HSCs ()
  5. Clinical trials of gene transfer into HSCs
  6. Chronic granulomatous disease
  7. Gaucher’s disease
  8. Fanconi’s anaemia
  9. Haemoglobinopathies
  10. Bibliography
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    Dunbar CE. Gene transfer to hematopoietic stem cells: implications for gene therapy of human disease. Annu Rev Med 1996; 47: 1120.
  • 3
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