Prospects for gene therapy using haemopoietic stem cells

Best Practice & Research Clinical Haematology Vol. 14, No. 4, pp. 823±834, 2001

doi:10.1053/beha.2001.0175, available online at http://www.idealibrary.com on

10 Prospects for gene therapy using haemopoietic stem cells Leslie J. Fairbairn*

PhD

Head of Cancer Research Campaign Gene Therapy Group

Joanne C. Ewing

BM BCh, BA Hons, MRCP, MRCPath

Cancer Research Campaign Clinical Research Fellow Paterson Institute for Cancer Research, Wilmslow Rd, Manchester, M20 4BX, UK

Gene therapy has thus far promised much and delivered little. Much of this has been due to de®ciencies in the reagents and methodologies employed in early clinical trials. Recent technological advances in vectors and haemopoietic stem cell manipulation, coupled with improved pre-clinical assays of gene transfer and expression in re-populating stem cells give cause for greater optimism. Here we review these advances and indicate areas requiring further development before clinical gene therapy in the haemopoietic system becomes a widely applicable treatment modality. Key words: gene therapy; pseudotyping; retroviral vector; cytokine; clinical trial.

Pluripotent haemopoietic stem cells (HSC) are an ideal target for gene therapy as they have the ability to self-renew and give rise to a life-long source of mature clonal progeny containing the transgene that may be expressed in all lineages. The rationale for this approach stems from the ability to treat genetic diseases successfully by the transplantation of genetically normal allogeneic cells whereby even in situations where mixed chimerism is achieved, with only limited contribution of normal donor cells to haemopoiesis, there is de®nitive correction of the phenotype.1 Until recently, transduction eciency into HSC has been low, with genes expressed in a relatively small proportion of cells. This practical limitation has con®ned the application of gene therapy to those candidate disorders where correction of a small number of cells may have a bene®cial e€ect. The spectrum of disorders amenable to treatment should be broadened by recent improvements in gene transfer technology. In order to achieve this goal the basic prerequisites for successful gene therapy include e€ective gene transfer and integration into the HSC genome with subsequent stable expression, while maintaining pluripotency and engraftment capacity. The seminal work on retroviral gene transfer methodology was performed in murine systems. The ability to achieve high-level stable transduction was promising and provided evidence of proof of principle. In contrast, initial attempts to recapitulate this *Principal correspondent. 1521±6926/01/040823‡12 $35.00/00

c 2001 Harcourt Publishers Ltd. *

824 L. J. Fairbairn and J. C. Ewing

work in large animal and human studies failed to achieve such ecient levels of gene transfer. The severe limitation on the number of engrafting HSC transduced resulted in only 0.1±1% gene marked cells in primates.2±5 The recent shift in focus from murine to human cell transduction has led to incremental technological improvements that, in combination, have led to the ability to transduce human HSC e€ectively, with levels of transduction detected at 5±20%.6±10 This has culminated in the recent report of clinically successful gene transfer with enduring engraftment of HSC engineered to express the gC cytokine receptor gene leading to correction of disease phenotype in two children su€ering from (SCID)-XI.11 It is clear that e€ective gene therapy in any system will be a function of the target cells, the eciency and control of gene transfer and expression, and the overall biological context in which transgene expression is to be achieved. Failure to address any of these adequately will lead to sub-optimal results on progression from pre-clinical models to clinical trial. Below we consider all of these elements and discuss the state of the art in approaching these along with further improvements that will be required before gene therapy becomes a widely available treatment option. DEFINING THE TARGET CELL The target cell for haemopoietic gene therapy may be derived from bone marrow, cytokine-mobilized peripheral blood stem cells (PBSC) or cord blood (CB). The HSC is de®ned by its ability to give rise to long-term multilineage reconstitution of an ablated host. It has become possible to purify, using cell-surface markers, a population of cells with high proliferative engraftment capacity. Immunomagnetic enrichment of CD34‡ progenitors is a standard procedure for isolating these cells for manipulation. While the population characterized by expression of CD34 and absence of di€erentiation antigens, such as CD38, is able to engraft, recent evidence shows that a CD34 ÿ , lineage-negative (linÿ ) cell is also capable of giving rise to long-term multilineage engraftment.12,13 This has been independently corroborated by the work of Goodell et al who demonstrated that long-term engrafting cells reside in the largely CD34ÿ/low side population (SP) cell fraction as de®ned by Hoechst 33342 dye.14 These ®ndings have led to concern that current strategies for stem cell isolation on the basis of CD34 may exclude other crucial engrafting cells.15,16 Notwithstanding this, CD34 selection remains the main means of de®ning the human HSC target of gene therapy. While reconstitution of haemopoiesis in a human host is the only de®nitive assay of HSC capacity, the need for pre-clinical studies necessitates the use of surrogate assays. In vitro assays, such as long-term culture-initiating cell (LTC-IC) assays, detect a primitive subpopulation of haemopoietic cells that include a minority with repopulating ability. However, gene transfer and expression in LTC-IC does not necessarily predict e€ective gene transfer and expression in stem cells capable of longterm reconstitution.17 For this reason, immune-de®cient animal models have found increasing importance in assessing gene transfer to human haemopoietic cells with repopulating capacity. By far the most information has been derived from the non-obese diabetic/severe combined immune de®ciency disease (Nod/SCID) mouse model and the recent b2-microglobulin-de®cient version of the model.18±21 The pluripotent stem cell that gives rise to multilineage human engraftment in this model is termed the SCID-re-populating cell (SRC) and represents a more primitive population than that de®ned using in vitro methodology. While far from perfect (many murine haemopoietic growth factors do not exhibit species cross-reactivity leading to a sub-optimal, cytokine-de®cient microenvironment in the xenograft), this is currently the most

Prospects for gene therapy 825

accurate predictor of clinical stem cell behaviour.17,22 Another xenograft system that provides a useful model is that using pre-immune fetal sheep, although time constraints and expense limit the utility of this model.21,23 TRANSDUCTION CONDITIONS With very few exceptions, gene transfer to primitive haemopoietic cells has been accomplished using the natural ability of viruses to infect and direct gene expression in these cells. While a number of di€erent viruses have been modi®ed to e€ect safe transfer of therapeutic genes, those based on retroviruses (oncoretroviruses and lentiviruses) remain the most important vectors for gene therapy in the haemopoietic system. Cell surface receptors An important factor determining the eciency of retroviral gene transfer is the availability of suitable receptors on the target cell, to which retroviral particles may bind and e€ect cell entry (Table 1). The majority of early pre-clinical and clinical studies have made use of the envelope from the amphotropic strain of murine leukaemia virus (MLV). While a great deal of useful information has been obtained with this envelope protein, it is now clear that the levels of expression of the amphotropic virus receptor on primitive human haemopoietic cells are low, compromising transduction eciency.24 For this reason a number of alternative envelopes have been used to pseudotype MLVbased retroviral particles targeting more abundant alternative receptors. Perhaps the most important of these is the envelope from the gibbon-ape leukaemia virus (GALV) which recognizes an alternative membrane protein, Pit-1, that is more widely expressed on primitive HSC.25 The use of this pseudotype has contributed to high levels of transduction of re-populating human and other primate cells.26,27 GALV is currently the pseudotype of choice for gene transfer into human HSC. Another pseudotype of note is vesicular stomatitis virus G-protein (VSV-G), a pantropic envelope whose cognate receptor is the ubiquitous phosholipid membrane component conferring a broad host range.28,29 This envelope protein shows greater structural stability which facilitates concentration of virions by ultracentrifugation, a step that leads to disruption of conventional retroviral particles. This allowed the use of very-high-titre retroviral Table 1. Pseudotyping strategies. Envelope

Receptor

Tropism

Packaging cells

Reference

Ecotropic

Neutral amino acid transporter

Rodents

GP ‡ E86, c-CRE

30 31

Amphotropic

Pit-2/Ram-1 phosphate transporter

Wide mammalian

GP ‡ envAm12, c-CRIP

32 31

GALV

Pit-1/Galvr-1 phosphate transporter

Wide mammalian

PG13

33

RD114

Sodium-dependent neutral amino acid transporter

Wide mammalian (not murine)

FLYRD18

34

VSV-G

Membrane phospholipid

Pantropic

GP7c-tTA-G10 293GP/tTAER/G

29 28

826 L. J. Fairbairn and J. C. Ewing

supernatant, reducing culture time to 1 day while preserving equivalent levels of transduction.6 Co-localization of target cell and vector The importance of the marrow stromal layer was recognized when culture on a matrix of autologous marrow ®broblasts improved levels of transduction using vector supernatant.35,36 The growth of adequate autologous stroma can, however, be problematic. The e€ectiveness of the stromal layer may be related to production of critical factors supporting cell survival combined with provision of co-localization domains for virus and stem cell. Subsequently it has been appreciated that the presence of a chymotryptic fragment of the extracellular matrix protein ®bronectin (recombinant CH-296) during ex vivo manipulation of stem cells signi®cantly increases the transduction of human haemopoietic cells through co-localization of vector particles and target cells.37,38 This has enabled a 10- to 50-fold increase in transduction eciency. There is also a possible in¯uence on cell viability and di€erentiation, preventing apoptosis and maintaining the regenerative functionality of the stem cell.27,39 Another approach to bring virus and target cell into close proximity has been centrifugation-mediated transduction or `spinoculation'. This has led to enhanced eciency of transduction.40 Induction of cell cycling During retroviral transduction, in order that the pre-integration nucleoprotein complex is able to gain access to the host cell chromatin there must be disruption of the nuclear membrane during progression through mitosis.41,42 HSC are, by nature, predominantly non-dividing, quiescent cells in the G0 phase of the cell cycle43 and thus relatively refractory to retroviral mediated gene transfer. The inclusion of cytokines during ex vivo transduction is critical for induction of cycling to promote retroviral integration and also to maintain the viability and pluripotentiality of primitive repopulating stem cells.44,45 Combinations of cytokines such as interleukin 3 and 6 (IL-3 and IL-6) and stem cell factor (SCF) were used in the early murine studies.46 This cytokine exposure is, however, detrimental to the re-populating capacity of HSC47±49, leading to loss of up to 40% of engraftment capacity in murine systems50,51, and an approximately fourfold decline in human SRC and primate reconstitution.6,52 For clinical gene therapy applications the de®nition of culture conditions which are able to support HSC proliferation without di€erentiation is essential. A plethora of studies have attempted to de®ne the key elements in maintaining totipotency and expansion using highly enriched populations of stem cells and cocktails of recombinant growth factors.53,54 In particular, the use of exogenous Flt-3 ligand is able to sustain clonogenicity of re-populating human cells, allowing extended ex vivo transduction.55 Thrombopoeitin has more recently been shown to have a potent ability to promote the viability and suppress apoptosis of HSC and is synergistic with Flt3 ligand.56 In combination, Flt-3 ligand, TPO, stem cell factor, IL-3, IL-6 and G-CSF are important synergistic factors with resultant transduction levels in SRC of 12±20%.57,58 Most of this work has focused on CB cells that may di€er in their cytokine requirements for promoting self-renewal divisions in comparison with adult marrow or PBSCs. We must be cautious regarding the generic applicability of these approaches to all sources of stem cells although reports of success are emerging to support these conditions in PBSC.10

Prospects for gene therapy 827

Another interesting approach has been to induce cell cycle entry through manipulation of the intracellular cyclin-dependent kinase (CDK) pathway. The assembly of the CDK/cyclin complexes that allow progression into cell cycle is checked by the presence of CDK inhibitors. Dao et al59 manipulated both p27kipÿ1 via antisense oligonucleotides and p15INK4B through a neutralizing antibody against transforming growth factor b (TGFb). They were able to show that the combined e€ects were to allow cell cycle progression in quiescent haemopoietic progenitors and demonstrated augmentation of the transduction of these cells while maintaining the ability to engraft immune de®cient mice. This acceleration into cell cycle may have the advantage of allowing transduction before the capacity for subsequent engraftment and lineage development is diminished. This strategy may point the way forward towards successful manipulation of the stem cell. As we understand more about the cell cycle and develop pharmacological agents to control cell cycling this may increase in importance. Priming of stem cells in vivo through systemic administration of the cytokines granulocyte-colony stimulating factor (G-CSF) and c-kit ligand has been shown to improve transduction of murine bone marrow cells.60,61 This approach has been extended and demonstrated in the rhesus macaque autologous transplantation model with recipients having up to 5% of circulating cells containing the vector a year posttransplant.62 The increasing clinical use of cytokine-mobilized peripheral blood stem cells for transplantation may make this a particularly appealing source of cells. Potential explanations for improved transduction eciency into in vivo primed cells include altered cell cycle status, and an e€ect on the expression of receptors for retroviral vector envelope proteins on the primitive stem cell.63,64 An alternative strategy to overcome the requirement for prolonged in vitro culture to induce cell cycling has been the development of vectors derived from human immunode®ciency virus type 1 (HIV-1). In contrast to type C retroviruses, the lentiviral pre-integration complex can enter the host nucleus without induction of cell division as proteins encoded by the viral genome allow active transport via the nuclear pore machinery obviating the requirement for an extended period of cell culture.65 Early evaluation of the lentivirus-based vectors has suggested that G0 cells remain somewhat resistant to viral integration and that cells must at least enter into cell cycle for e€ective transduction.66,67 This may not present a problem practically, as the intracellular viral viability is prolonged and integration may occur following transplantation and homing as cell division occurs in vivo in the bone marrow microenvironment.66 There is as yet no evidence to support any increase in lentiviral transduction eciency compared with optimized ex vivo manipulation and transduction based on murine retroviral vectors.67,68 EXPRESSION Early retroviral vectors were based on Moloney murine leukaemia virus (MoMuLV). Such vectors have shown utility in pre-clinical models of gene therapy and have been utilized in early and recent clinical trials. While there are reports of long-term gene expression from such vectors in human subjects, it is also evident that transgene expression may be subject to down-modulation (i.e. silencing) and to positional variegation.69 At least part of this e€ect is due to a lack of appropriate transcription factors in stem cells. However, a number of inhibitory cis-acting elements, mapping to the viral enhancer and primer binding site in MoMuLV, have also been shown to a€ect expression in stem cells negatively.70±72 Thus, signi®cant e€ort has recently gone into

828 L. J. Fairbairn and J. C. Ewing

the development of alternative vectors, utilizing elements that may confer more optimal expression in stem cells. Ostertag and colleagues de®ned a novel vector, termed murine embryonic stem cell virus (MESV), which carried a number of point mutations in the LTR and an alternative primer binding site.73 This vector showed enhanced expression in murine embryonic stem cells and, based on this, Hawley and colleagues developed the murine stem cell virus (MSCV) vector. MSCV is very ecient at directing persistent transgene expression in human re-populating cells transplanted into Nod/SCID recipient mice.74,75 Furthermore, in a primate model, stable in vivo expression of a marker gene was observed over a period of 6 months.75 Baum and colleagues have combined the primer-binding site of MESV with LTR sequences containing the enhancer/promoter region of murine spleen focus-forming virus (SFFV).76 The SFFV enhancer/promoter is eciently expressed in Nod/SCID repopulating human CD34‡ cells and their progeny with maintenance of expression documented for up to 4 months in this model.77 Both MSCV and SFFV are strong candidates for clinically applicable vectors, and the results of human trials with these vectors are awaited with interest. In the trials alluded to above, it is still expected that gene expression in a proportion of stem cells and their progeny will be attenuated±due partly to positional e€ects of the chromatin organization leading to sequestration of vector to transcriptionally inactive chromatin domains. Work identifying and testing stabilizing elements such as insulators and sca€old attachment regions may lead to more consistent and positionindependent gene expression from future vectors. Similarly, a greater understanding of the post-transcriptional events governing transgene expression is likely to lead to enhanced levels of transgene product. It should be noted that the promoter/enhancer combinations utilized in vectors undergoing or approaching clinical trial are still relatively transcriptionally promiscuous. While this may not be an important factor in some applications, for others, such as haemoglobinopathies or thalassaemias, it may be either desirable or necessary to restrict gene expression to a speci®c haemopoietic lineage. Thus some e€ort is being put into the identi®cation of lineage-restricted promoter/enhancer combinations.78 As gene therapy in the haemopoietic system progresses conceptually and practically, it is likely that tailor-made vectors, incorporating a wide range of control elements to achieve ecient, position-independent, lineage directed and perhaps homeostatically controlled expression will be required. HOST FACTORS CONTRIBUTING TO SUCCESS OF GENE THERAPY APPROACHES The greatest cause for optimism in HSC gene therapy has come from the report of a successful clinical trial in patients with (SCID)-X1.11 Part of this success owes much to recent advances in gene therapy technology discussed above. However, two important aspects of the host environment are also worth considering. First, the recipients are severely immunode®cient, thereby limiting immunological rejection of infused cells. Second, genetically corrected T-cells have a survival and proliferative advantage over their uncorrected counterparts. Attenuation of transgene expression has been reported in a number of animal studies, correlating with an immunological response to transgene products even in the face of immunosuppressive conditioning.79 Similar reactions to viral and marker genes have been seen in human studies80 and we have observed a humoral response to an

Prospects for gene therapy 829

endogenous human protein in a clinical trial of mucopolysaccharidosis type I (unpublished data). This is hardly surprising because transgene expression in the progeny of HSCs will inevitably lead to expression in professional antigen-presenting cells. As the technological issues surrounding gene transfer and expression in repopulating human haemopoietic stem cells are resolved the issue of graft/host interactions, particularly immune responses, will come to the fore. The current progress in gene transfer to mesenchymal stem cells, which exhibit low immunogenicity and can actively suppress T cell responses, may a€ord an option for tolerization. It is now understood that cells manipulated ex vivo have an engraftment disadvantage compared with unmanipulated cells.6,50±52 Further to this, stem cells that undergo cell division ex vivo (and hence become retrovirally transduced) may be further compromised compared to their ex vivo manipulated, untransduced counterparts. The e€ect of this would be to severely disadvantage gene-modi®ed stem cells in the competitive re-population phase following transplantation, leading to low levels of engraftment of gene-modi®ed cells. While this may be minimized with the use of optimized culture conditions, one further strategy to circumvent this might be to provide a selective advantage, in vivo, to gene-corrected cells. With the exception of a few, rare, examples (including SCID-X1 and ADA-SCID), the therapeutic transgene is unlikely to provide such an advantage per se. One approach is to provide a second, selectable, gene in cis with the therapeutic sequence. Notable examples include genes such as dihydrofolate reductase, O6-alkylguanineDNA-alkyltranferase and MDR-1 which confer resistance to cytotoxic drugs.81 An important consideration will be whether selective agents need to be administered acutely (which may pose only a limited additional risk over pre-transplant conditioning) or chronically. If the latter ensues, then the attendant toxicity of such selection may limit the ecacy of this approach. An alternative may be to provide an inherent survival or proliferative advantage to gene modi®ed cells. The use of Bcl family members as survival factors82, HOX-B4 as a regulator of early haemopoietic cell proliferation83, or of modi®ed cell surface receptors facilitating inducible proliferation84, may be applicable in this respect. However, as with selection of drug-resistant populations, the long-term sequelae of such approaches will need careful analysis.

PROCEEDING TO CLINICAL TRIALS As pre-clinical studies progress to clinical trials a new set of considerations will come to the fore. The use of gene therapy strategies, as with any other novel therapy, must be of minimal risk to patients. Potential adverse outcomes relate to problems associated with the autograft procedure and those speci®c to the gene therapy strategy, and may include emergence of competent retrovirus, insertional mutagenesis, failure of engraftment of manipulated cells, in¯ammatory responses and introduction of infection during in vitro manipulation. These potential risks have to be balanced against currently available therapies, as well as the often considerable morbidity and mortality related to the genetic disorder itself. Rigorous quality assurance of cellular manipulation and manufacture of large volumes of vector will be paramount and the development of closed systems in a similar approach to that undertaken in blood banking practice will aid the safe delivery of this therapy to the clinic. In view of the requirement for longterm follow-up, including testing for emergence of competent retrovirus and ultimate

830 L. J. Fairbairn and J. C. Ewing

longevity of transplanted cells able to express the transgene, gene therapy trials will be best conducted where there are facilities to ful®l this. SUMMARY The progression from pre-clinical attempts at e€ective transduction of the human haemopoietic stem cell to the ®rst report of successful correction using such manipulated cells has been dependent on a more coherent understanding of human stem cell biology allowing a series of incremental developments in the ®eld. These include engineering of novel vector systems for stem cell gene transfer, targeting the cell surface to e€ect entry and minimizing elements that can lead to silencing of expression in the di€erentiated progeny. Another key development has been the re®nement of conditions for ex vivo manipulation which maintain integrity and functionality of the engrafting pluripotent stem cell while creating an environment which enables retroviral integration through induction of cell division. Advances in surrogate in vivo assay systems have allowed the rapid optimization and evaluation of these clinically applicable protocols. All of these in combination with strategies to facilitate in vivo selection are expected to lead to increased success in clinical gene therapy using haematopoietic stem cells. Currently, the demands of gene therapy are relatively simple, i.e. ecient transfer and expression of a transgene; however, as the clinical application of gene therapy becomes a reality, inevitably a more adaptable and precisely controlled expression system may become applicable clinically. Practice points Optimal integrated gene transfer protocol in order to achieve maximally ecient haemopoietic stem cell transduction: . target cell: CD34‡ cell selected from cytokine-mobilized peripheral blood/ marrow or cord blood . pseudotype: GALV . co-localization: ®bronectin fragment CH-296 . cytokines: Flt3 ligand, thrombopoietin, stem cell factor, IL-6+G-CSF . vector: SFFV or MSCV . host factors: selective pressure toward donor cell engraftment/ survival

Research agenda Clinical . determine engraftment and expression levels in clinical setting . determine longevity of expression and engraftment . determine practicalities of in vivo selection Pre-clinical . further development of lentiviral systems . further develop vectors to allow lineage-directed and controlled expression . improve pharmacological agents for in vitro manipulation of the stem cell

Prospects for gene therapy 831

Acknowledgements JE is funded as a Clinical Research Fellow by the Cancer Research Campaign.

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