Gene transfer into human haematopoietic stem cells

Pergamon

Transfus. Sci. Vol. 18, No. 2, pp. 291-311, 1997 Copyright © 1997 ElsevierScience Ltd. All rights reserved Printed in Great Britain PH: S0955-3886(97)00021-0 0955-3886/97 $17.00 + 0.00

Gene Transfer into Human Haematopoietic Stem Cells Bruno P6ault, PhD* Pierre Charbord, MD't:t:

• This review of gene transfer to the human haematopoietic system (1) describes the different vectors used to transduce genes into stem cells, emphasizing retroviruses that have already shown their efficiency and innocuousness; (2) analyses which human cells should be targeted to ensure long-lasring engraftment; (31 indicates the different means of infecting these targets ex vivo, underscoring the role of cytokines and stromal cells; (4) recollects the methods used to evaluate transduction efficiency; and (5) gathers the results of clinical trials recently performed using human stem cells. The major conclusions are that good practice can ensure safe gene delivery to human beings and that longlasting, multilineal precursors can be transduced using retroviral vectors of marker genes or genes of therapeutic interest. However, transduction rates appear to remain relatively low, which should stimulate ongoing research on both vector design and means of e x vivo gene transfer. © 1997 Elsevier Science Ltd •

INTRODUCTION In 1980, Cline e t al. 1 reported the first successful gene transfer experiment in intact animals. The target tissue was mouse bone marrow that was treated with calcium precipitates of DNA enriched in dihydrofolate reductase sequences, and the outcome was partial methotrexate resistance in recipients of the genetically modified cells. The authors concluded that, in addition to drug resistance induction, "... hemoglobinopathies ... seem to be natural targets for treatments by gene transfer techniques". That prescient opinion has obviously gained widespread acceptance. The list of diseases--haematological or not--which are candidates for gene therapy, has lengthened as a result of progress made in molecular biology and pathology, which has converged with the development of improved vectors to bring gene transfer into the clinic. Again, haematopoietic cells were first chosen since they are accessible for e x v i v o manipulation and easily engrafted. In 1990, Blaese e t al. 2 transplanted autologous peripheral blood leukocytes modified by retroviral transfer of the adenosine deaminase [ADA) gene into children suffering inherited severe combined immunodeficiency [SCIDI. In the meantime, characterization of the human haematopoietic cell hierarchy has been much improved and there exists the possibility that widespread and long-term dissemination of a

"Institut d'Embryologie Cellulaire et Mol6culaire du Coll~ge de France et du CNRS, 94736 Nogent-sur-Mame, France ~'Laboratoire d'Etude de l'H6matopoi~se, Etablissement de Transfusion Sanguine, 1 bvd A. Fleming, 25000 Besan~on, France. **Author for correspondence. Fax: 33 3 81 61 56 17. 291

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therapeutic transgene can be achieved after transduction into stem cells. Gene transfer experiments in the well-characterized and easily manipulated haematopoietic system of mice have provided the theoretical basis for human gene therapy. However, the use of larger mammals and, eventually, human trials has revealed many problems in ensuring sufficient, long-term transgene expression in a clinical setting. Extensive basic research has long been carried out in the areas of vector design, gene transfer techniques and cell targeting, the progress of which has already been reviewed in a number of successive articles, a-8 We would like to briefly summarize the current status of experimental and clinical gene transfer into human haematopoietic cells now that the long-term engraftment in human patients of transgenic, multipotential precursor cells has, at last, been documented. 9-~2

VECTORS FOR GENE TRANSFER

Naked DNA molecules cannot spontaneously penetrate into cells, and the accomplishment of gene therapies relies on the development of efficient, convenient and safe gene delivery systems. Physical techniques used at the bench to experimentally transfer DNA into cells, such as electroporation, calcium phosphate precipitation, "gene gun" shooting or intranuclear microinjection are usually not considered for gene therapy application as they all suffer one or more major limitation including low frequency of modified cells, the need for exceedingly large amounts of DNA, high cost, significant target cell toxicity and lack of chromosomal integration. As a recent exception, however, the transfer of a viral replication-inhibiting transdominant r e v mutant via plasmid-coated gold particles into CD4+ T cells has been reported to prolong target cell survival in HIV-infected individuals, la Such nonintegrative gene transfer obviously

could not be applied to stem cell-mediated gene therapy. For the same reason, cationic liposomes, which can infect a broad range of cells 14 and carry hope for the gene therapy of differentiated cells in tissues such as the respiratory tract, do not represent, in their present state of development, serious candidates for gene transfer into stem cells. Since the original report of foreign gene transfer with a retrovirus into mouse haematopoietic cells) s retroviruses represent the vectors of choice for sustained transgenesis in actively renewing tissues such as the haematopoietic system. Replication-defective retroviral vectors like those derived from the Moloney murine leukaemia virus {MoMuLVJ are produced by replacing the gag, pol and e n v viral genes by the sequence(s} to be transferred up to about 5 kb in size--under the transcriptional control of viral long terminal repeats ILTR} or of internal promoters. Transcribed viral genomic RNAs are then "packaged" into infectious viral particles following transfection into an established mouse fibroblast cell line engineered to include the "helper" Moloney gag, pol and e n v genes, but not the cis-acting sequences necessary for packaging, which, in contrast, are intact in the transfected recombinant sequence. By complementation, the full-length recombinant vector RNA genome is therefore packaged with the products of the helper genome. The produced viruses can infect cells, leading to random integration inside their genome of vector sequences that, however, are unable to go through another cycle of replication in the absence of intrinsic helper sequences Ireviewed in refs 6-8,16 and 17). Most currently used retroviral vectors for experimental and clinical gene transfer into human haematopoietic cells are amphotropic MoMuLV-derived mouse viruses. 4 While MoMuLVs normally infect mouse cells only, these vector viruses can also infect human cells because they have been engineered to harbour the envelope e n v gene of a

Gene TransIer into Stem Ceils

unique murine leukaemia virus with amphotropic range. The use of such recombinant retroviruses to transfer therapeutic genes in humans also relied on the initial demonstration of their lack of pathogenicity in primates, is,19 However, monkeys engrafted with cells infected with vector preparations contaminated with replication-competent virus developed aggressive T-cell lymphomas, 2° an observation that stressed the importance of stringently screening retrovirus-producing clones for the presence of helper virus. Currently used packaging cell lines virtually guarantee the absence of replication-competent particles. MoMuLV-derived, and notably MFG vectors, ensure relatively efficient transgene expression in haematopoietic cells, 21,22 likely due to high levels of spliced RNA synthesis. ~3 Further improvements might be achieved with stem cell-targeting vectors that would include regulatory elements mediating, for example, CD34 expression, 24 while lineage-specific promoters might be useful for driving transgene expression, preferentially, along one avenue of haematopoietic differentiation. ~s From a more practical point of view, the addition to the therapeutic sequence of a marker gene encoding a conveniently detectable cell surface protein would permit the selection, prior to engraftment, of transgenic precursor cells by flow cytometry. 26 Although they have been used in virtually all gene therapy attempts with the h u m a n haematopoietic system so far, amphotropic mouse retroviruses now have challengers. Because its receptors are abundant on human bone marrow cells, the Gibbon Ape Leukaemia Virus (GALV) offers promising prospectives for gene transfer into haematopoietic cells. Producer cell lines for the GALV envelope could be used to package murine recombinant retrovirus e s y or, alternatively, murine retroviral vectors could be pseudotyped with the GALV envelope28 in order to increase infectivity.

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Adenovirus vectors do not integrate into target cell chromosomes and therefore cannot be envisioned for stem cellmediated gene therapy but could be used to transfer therapeutic genes to be expressed transiently in haematopoietic cells, such as cytokine genes in cancer. 29-al In contrast, the small DNAcontaining adeno-associated viruses (AAVs) are attractive vector systems (reviewed in ref. 32) since they can infect a broad range of target cells and-at least for wild-type AAVs--integrate stably and predominantly at a specific site in chromosome 19, which makes it less likely that they can induce insertional mutagenesis, and may guarantee consistent levels of transgene expression (reviewed in ref. 7), in the absence of any known pathogenic effect. Interestingly, AAV seems to infect immature haematopoietic precursor cells in the absence of cytokine stimulation) a-as A major drawback, however, is that AAVs are dependent parvoviruses needing helper viruses--usually adenoviruses-to replicate. In the laboratory, recombinant AAVs are cotransfected with helper adenoviruses into cell lines from which infectious AAV vectors are retrieved after lysis. These laborious protocols are not appropriate to gene therapy and the use of AAVs in the clinic will have to await the development of efficient helper cell lines. Several other virus species, notably HIV-1, 7,a6 have been considered for engineering into gene transfer vehicles for blood cells. Their description is, however, beyond the scope of the present review. All in all, and despite extensive research and development of alternative systems, the vast majority of ongoing or planned clinical trials on the gene marking or therapy of the human haematopoietic system make use of relatively simple, monocistronic recombinant amphotropic murine retroviral vectors that, at the moment, represent the best compromises between infection efficiency, duration of transgene expression, tolerability by the organism and innocuousness.

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HAEMATOPOIETIC TISSUE TARGETS Extensive renewal capacity and relatively easy accessibility for e x v i v o manipulation contribute to making blood tissue a privileged candidate for gene-mediated therapy. Theoretically, this can be achieved permanently either by the repeated correction of mature cells or their immediate precursors or, more satisfactorily, by unique gene transplantation into the most primitive haematopoietic stem cells. Adult bone marrow remains the most widely used reservoir of therapeutic haematopoietic cells, yet other sources of blood-forming cells are contemplated or have already proven useful for clinical use. Allogeneic fetal liver has been transplanted into infant patients, and even in utero, to treat inbom immunodeflciencies and errors of metabolism [reviewed in ref. 37); however, harvesting autologous fetal liver cells for genetic modification and engraftment in utero has not been reported. In an alternative protocol, retrovirally-conveyed genes administered by direct intrahepatic injection to rat fetuses were detected by PCR and Southern blot analysis until 26 weeks post-natal, albeit in low numbers, in the bone marrow and other haematopoietic tissues, as This experiment models direct in utero gene therapy, a potentially interesting approach in the future. More realistically, currently, fetal blood cells harvested in transplantable amounts from the umbilical cord at birth and used to treat haematologic diseases in young allogenic human beings (reviewed in ref. 39) could prove invaluable, following genetic modification and autologous engraftment, for the therapy of disorders diagnosed during pregnancy. Cord blood precursor cells, including those recovered after cryopreservation, can be efficiently infected with recombinant retroviruses 4°'~ and transgene integration and expression can be traced until their differentiated T-cell progeny, 44,4s sug-

gesting protocols for the therapy of inherited or in utero acquired immunodeficiencies. Indeed, neonates suffering SCID due to the absence of ADA have been transplanted with their own cord blood precursor cells in which ADA cDNA had been retrovirally transduced.12 The numerous haematopoietic stem ceils that are recruited in circulating blood by chemotherapy and/or growth factor infusion can be repeatedly harvested by apheresis and can ensure both short-term and long-term haematopoietic recovery after autotransplantation. These cells therefore represent coveted targets for therapeutic gene transfer. Mobilized peripheral blood cells {PBMC) are at least as susceptible to infection with recombinant retroviruses as their bone marrow counterparts. 11,46,4z When cotransplanted with distinctly retrovirally marked autologous bone marrow cells into patients, transgenic PBMC even seemed to contribute to longer term engraftment of multiple cell lineages. 11

METHODS OF VIRAL INFECTION EX VIVO

As already alluded to in the previous section, to obtain protracted results one should aim for the most immature precursors, those able to reconstitute the host for long periods of time. Studies on the haematopoietic stem cell hierarchy (reviewed in ref. 48) in the mouse indicate that these cells are probably different from more mature precursors responsible for short-term reconstitution, by a number of parameters including the expression of cell surface antigens, the location in the cycle and the ability to grow in vitro. The fact that reconstitution appears to occur in two waves, an early one (months following grafting} when numerous relatively mature precursors contribute to haematopoiesis and a later one (years following grafting) when few immature precursors are involved, suggests that, if one is looking for early and late expression of

Gene Transfer into Stem Cells

genetically-modified cells, which is the case in a clinical setting, one should infect both early and late precursors, i.e. a fairly broad spectrum of cells. In other words, the target cells should n o t be a highly selected population. In humans, early and late precursors bear the CD34 membrane antigen; CD34+ cells appear therefore as the target population of choice. However, mature CD34+ cells are actively cycling while immature cells are out of cycle. Since retroviruses are integrated only in actively cycling cells one should induce immature cells residing in Go to enter into cycle for a few rounds of division before returning out of cycle. Here resides the actual quandary since there are no experimental data indicating that this procedure is possible. On the contrary, during haematopoiesis, proliferation and differentiation are tightly coupled. One has to assume that uncoupling is possible at the stem cell level. Experiments on transduced cells should actually indicate whether this is possible and what experimental procedure has to be used to achieve this aim. Actual experimental procedures have used three types of target cells: unseparated bone marrow cells, mononuclear cells and CD34+ cells. Unseparated bone marrow cells from dogs were cultured using a longterm culture system by Carter e t al. in 1992. 49 It has been well known since the early eighties that, in long-term marrow culture, microenvironmental cells are generated in the adherent layer. These cells, comprising macrophages and non-haematopoietic stromal cells, ensure the maintenance of immature precursors for long periods of time. In the murine system for example, cells with erythropoietic competitive repopulating ability can be recovered for several months from this layer. It has been shown that the maintenance of "stem ceils" was under the tight control of the microenvironmental cells, which are able in particular to secrete growth factors after weekly medium renewal. These factors allow the entry of immature

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precursors into the cycle ~ on the contrary, inhibitors, also derived from microenvironmental cells, would take over 3-4 days after m e d i u m change, which would allow the "stem cells" to re-enter Go. This modulation of the stem cell cycle appears therefore to be optimal for transduction experiments. Carter e t al. 49 supplemented the culture m e d i u m on days 0, 7 and 14 with viral supernatant containing 106pfu mL -1. Non-adherent and adherent ceils were collected at day 21 and infused in conditioned and non-conditioned dogs. Transduced progenitors and T ceils were detected by PCR up to 20 months after grafting and progenitors expressing the marker gene at the protein level were detected for about 2 years after grafting, indicating long-term reconstitution. Moreover, the study of granulocyte macrophage colony-forming units (CFU-GM) expressing the marker gene confirmed the two waves of reconstitution with the highest number of progenitors seen within the first 6 weeks. Finally, this work suggested that a marrow-ablative regimen was not mandatory to ensure stem cell grafting. These data confirmed the interest of using long-term culture systems for stem cell transduction and were recently applied to h u m a n marrows from normal individuals and patients with Hurler's syndrome (a-L-iduronidase deficiency),s° Viral supematants {constructs with u-Liduronidase under LTR promoterl were added once daily for 4 days after culture inception. Although a-L-iduronidase was not detected in the supematant from cultures of normal individuals, 10 nmol h - t 10-6 cells were detected in transduced cells for up to 18 weeks. In culture supematant from patients with Hurler's syndrome whose cells were transduced, levels reached up to 100 nmol h -110 -6 cells for an identical culture time. A m a x i m u m of 60% of the progenitors collected from week 9 cultures contained the vector and adherent macrophages, showing glycosaminoglycan deposits in non-transduced cultures from patients, recovered a normal

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appearance in the transduced cells. In this work the addition of growth factors to the culture medium did not improve the results. These studies in large organisms, dogs and humans, clearly indicate that the use of long-term culture systems is efficient for transducing reconstitutive cells, both early and also in late reconstitution. This system appears, therefore, very attractive. However, our own experience with long-term marrow culture in patients suggests a potential problem, that of insufficient stromal layer formation. In this case, the overall cell recovery after three weeks [i.e. a culture duration sufficient to allow the entry into cycle of immature precursors} may be insufficient and the grafting ineffective. This is why the use of preformed adherent layers, as discussed below, is an appealing alternative. For transduction studies some authors have used mononuclear cells from marrow separated using discontinuous density gradients [usually flcollhypaque). Such procedures have the advantage of simplicity, remove mature red cells and polymorphs and allow the study of the effect of accessory cells, such as monocytes and lymphocytes. However, it has been shown by yon Kalle et aL 2s that enrichment in CD34+ cells resulted in more efficient transduction than when whole mononuclear cells were used [the end point being the analysis of gene expression in CFUGM}, which may be due to removal of cells that have a higher vector affinity than progenitors, to increased progenitor cell cycling after removal of endstage leukocytes or to increased multiplicity of infection for CD34+ target cells. In addition, numerous studies have shown that the significant accessory population is the stromal cells. Presently, it appears safe to consider the optimal target cell population to be CD34+ ceils from bone marrow, peripheral blood or cord blood taken as a bulk cell suspension or as single cells, although, as underlined previously, it

may be of theoretical interest to consider whether more immature subsets are transduced as well. Whatever the means of infection chosen, two parameters have to be considered: the availability of the infectious units and the state of responsiveness of the target cells. Infectious units may be provided directly by the packaging cell line, target cells being seeded on the line for variable spans of time, or added as viral supernatant; in the latter case, although the highest possible viral titre appears, for most, to be desirable, 19,s~ for some investigators its significance still remains a matter of debate, s2,s3 Responsiveness of the target cells implies that these present membrane amphotrophic receptors and are able to integrate the virus, i.e. are actively cycling. It has been recently shown that mRNAs for amphotropic receptors in CD34+ or CD34+/38- marrow cells may be increased by incubating the cells for 60 h with growth factors and in particular with 10ng mL -1 interleukin ;3. 54 Growth factors are also effective in inducing the entry into cycle of resting precursors. Several growth factors have been used before and during incubation of precursors with viral supernatant~ some examples of such cultures are shown in Table 1. Any conclusion as to the best cytokine combination is difficult to reach since the protocols are highly variable: variability of the target cells Imononuclear vs CD34+ cells, cells from bone marrow, from peripheral blood, collected under stationary conditions in normal subjects or after chemotherapy and infusion of cytokines in patients, and cells from cord blood), infection in the presence or absence of the packaging cell line, cytokine addition before or simultaneously with the addition of viral supernatant, variation in the duration of infection. However, one may try to extract some general rules from the data. The association of IL-3, IL-6 and SCF is found in many protocols and has been used in clinical studies~l 1,12,62 concentrations are usually high [10-100ng mL-1). Some

Gene T r a n s | e r into Stem Cells

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Gene Trans|er into Stem Cells

other cytokines may be of interest for specific target cells: epo for cord blood precursors, 41 bFGF for peripheral blood precursors under stationary conditions, 6° as opposed to mobilized cells after chemotherapy and cytokine infusion when addition of growth factors i n v i t r o would be irrelevant. 46 A recent work by Kn/ian-Schanzer et al. 6a indicates limitation for the use of cytokines, showing that CD34+/lineage+ marrow ceils enter into cycle after cytokine addition and that a subset of these is therefore regularly transduced in contrast to more primitive CD34+/lineagecells, where the entry into cycle is highly variable. This probably explains why few cells, if any, are transduced. Finally, Hatzfeld e t al. 64 have recently proposed another strategy that may prove of interest in the future: the use of antisense agents, blocking the effect of negative regulators {in this case TGF/~ll produced by the precursors in an autocrine way and fettering active cell cycling. Stromal cells, serving as a feeder layer during the transduction procedure, have been used in several studies. Examples of studies in large mammals {dogs, non h u m a n and human primates} are shown in Table 2. Here again there are many differences between studies. However, the reasonable conclusion that stromal cells are beneficial may be drawn. Remaining questions concern the nature of the stroma, whether cytokines have to be added, the length of the coculture and the frequency of viral particle addition. Most investigators have used allogenic irradiated primary layers of long-term marrow cultures. However, Nolta e t aL 6s have shown that the instrumental cells are stromal ceils and not macrophages. These cells probably correspond to marrow myofibroblasts. The negative results obtained by Xu e t aI. 61 when separating CD34+ ceils from stromal cells by an insert I"non-contact culture" using Transwell plates} suggest that haematopoietic precursors have to be in contact with the stroma, as opposed to results in the

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mouse, where efficient transduction has been obtained using a "non contact" system. 71 Whether cytokines have to be added to the coculture of stromal cells and haematopoietic precursors may depend on the nature of the precursors, as suggested by Nolta e t al. 68 showing that cytokine addition was irrelevant to transduce bone marrow CFU-GM, as opposed to peripheral blood cells. However, Xu e t al. 69 refuted this result, indicating better efficiency when IL-3, IL-6 and SCF were added, whatever the origin of the CD34+ ceils. As already discussed, cytokine amounts and combinations are probably of crucial importance. The length of coculture is usually brief {less than 1 week}. However, von Kalle e t al. 28 obtained optimal results with a coculture time of 3 weeks. This latter parameter is probably linked to the amount and frequency of addition of viral particles. It has been recently demonstrated 72 that a protocol using bone marrow CD34+ cells cocultured for 3 days with passaged stromal ceils, growth factors {IL-3 + IL-6 + SCF} and viral particles {being added once daily}, allowed the efficient transduction of multipotential {myeloid and lymphoid} precursors as evidenced after 11 months in b n x mice. This result clearly indicates that, under these conditions, transduction of very primitive human cells {with the characteristics of "stem cells"} is possible, which is of great theoretical importance. However, implementation in a clinical setting may be cumbersome due to the growth of adequate stroma prior to the transduction procedure. The role of stromal cells may be explained in many different ways. Stromal cells express many cytokines; the relatively negative results obtained by Moritz, Keller and Willliams 4° using human bone marrow precursors grown on murine stroma engineered to express the transmembrane form of SCF {220 kDa in molecular weight due to the splicing out of exon 6J suggest that it may be premature to favour the role of a specific cytokine. Stromal cells also

300 Transfus. Sci. Vol. 18, No. 2

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Gene Transfer into Stem Cells 301

express cell adhesion molecules either on their membrane or exported in domains of extracellular matrix glycoproteins. The work of Moritz, Patel and Willliams 7a is interesting in that respect. These authors reported sarisfactory gene transfer into CFU-GM or long-term culture initiating cells {LTCIC), when light-density non adherent human marrow cells were seeded, after stimulation with IL-6 and SCF, on fibronectin-coated plates. Moreover, they showed that the major heparinbinding domain lincluding the CS-1 region) could substitute for the whole fibronectin molecule za by binding both viral particles and target cells. TM This result has been recently confirmed by Freie e t al. 7s using CD34+ cells from cord blood. In conclusion, at the present time there appear to be many parameters involved in the successful transduction of haematopoietic precursors. Clinical protocols will be the ultimate test for several allegedly critical parameters. The reader is referred to the exemplary recent clinical protocol set up by several U.S. teams z6 concerning "the retroviral mediated transfer of the cDNA for human glucocerebrosidase into haematopoietic stem cells of patients with Gaucher Disease" that will test several parameters for e x v J v o transduction.

THE M E A N S OF E V A L U A T I O N

One should evaluate whether the gene of interest has been integrated into the genome of, and is expressed {at the mRNA and protein levels) by, the appropriate target cell. There are two types of inserted genes: marker genes that should not modify the behavior of the cell target, and therapeutic genes that should be actively expressed in the cell and modify its behaviour and that of its progeny. The most widely used marker gene is the bacterial neomycin phosphotransferase gene conferring resistance to neomycine {neog}. Some studies have also used the bacterial lacZ

or nls lacZ genes. Integration of the gene is proven by studies at the DNA level [Southern blotting or PCRJ. Expression is proven by studies at the mRNA level Creverse-transcriptase PCR} or, more usually, at the protein level. Cells expressing neoR survive in the presence of the neomycin analogue, G 418. Cells expressing lacZ or nls lacZ express bacterial neutral/3-galactosidase in the nucleus and cytoplasm or in the nucleus only. As stated earlier, marker genes should not affect the target cell behaviour. However, it has been reported that expression of neoR gene in NIH3T3 cells may modify the glucose catalysis pathway 77 and we have recently observed a lesser expression of aSM actin in murine marrow stromal cells infected with a neoR/lacZ retroviral vector. Similar minor alterations of cell behaviour have not yet been reported for transduced haematopoietic precursors. In a more convenient and quantitative approach, gene transduction targets within, for example, subsets of the CD34+ cell population can be directly characterized by multicolour FACS analysis following transfer of genes encoding cell membrane antigens Iselected or engineered to avoid any modification of the behaviour of the transduced celll. Twenty-four hours after retroviral transfer of the heatstable antigen (HSAI gene into immature bone marrow cells, Conneally e t al. TM detected HSA expression at the surface of about 30% of the analyzed CD34+CD38- cells. Similar observations were recently made by Champseix e t al. {unpublished datal, who could see expression of the mouse CD2 antigen ImCD21 at the surface of CD88-CD34+ and Thy-l+CD34+ immature cord blood haematopoietic cells 3-4 days after retrovirus-mediated mCD2 gene transfer in the CD34+ cell population. If confirmed, these results would provide direct evidence for gene transduction into very primitive CD34+ precursors. Genes of therapeutic interest are very diverse. So far, investigators have tried to transduce the following genes:

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glucocerebrosidase, 61,69,79 deficient in Gaucher disease, ADA 12'19'55'58,62,67'80 whose deficiency is responsible for approximately one quarter of the cases of SCID, Fanconi anemia type C complementation gene, 43 not expressed in approx. 10-15% of the patients with Fanconi anemia, ~L iduronidase, s° deftcient in mucopolysaccharidosis type I, the most severe clinical presentation of which is Hurler's syndrome, gp 91phox, s9,7° deficient in patients with the X-linked form of chronic granulomatous disease, CD18, sl,s2 defective in leukocyte adhesion deficiency syndrome, multiple drug resistance gene, sa whose increased expression in haematopoietic precursors would allow more intensive chemotherapy, and mutant dihydrofolate reductase, 6s conferring resistance to methotrexate. As for marker genes, gene integration has been checked at the D N A level and gene expression at the mRNA and protein levels (including measurement of enzyme concentration); modification of cell behaviour has been checked at the cell level: for example, return to normalcy of cultured macrophages from patients with Hurler's syndrome5° or reduction of nitrobluetetrazolium by cells from patients with chronic granulomatous disease, s9 Whether the gene of interest is expressed in haematopoietic precursors can be checked using in vitro assays and in vivo examinations. Two in vitro assays are widely used: the growth of clonogenic progenitors in semi-solid medium and clonogenic more immature precursors in stromal cell-based cultures. The more widely used stromal-cell based assay is that for LTC-IC, where haematopoietic precursors are seeded at limiting dilution on an irradiated adherent layer from longterm marrow culture and secondarily {after 5 weeks) seeded in methylcellulose to evaluate their capacity to generate progenitors. Whatever the precursors assayed, the presence of the retroviral vectors in target cell genome may be checked by PCR using colonies plucked individually and their expression may

be assessed using RT-PCR on individual colonies, by growing colonies in the presence of G 418 {in the case of neoR), or by staining colonies with XGal (in the case of lacZ or nls-lacZ}. Recently, an inverse PCR method applied on cells from individual colonies has allowed determination of whether the retroviral integration site was identical from one colony to the other, z2 The biology of haematopoietic stern cells has been deciphered in the living mouse in dynamic models of blood cell ablation/reconstitution [reviewed in ref. 84) for which no surrogate in vitro systems are yet available. That major limitation for the characterization of h u m a n stem cells has been partly circumvented by the development of xenochimera models in which human haematopoietic tissues and cells are transplanted into immunodeficient and, hence, tolerant hosts. SCID or Beige/ Nude/Xid {bnx} mice can support, for several months, the maintenance of multilineage myeloid progenitors and the development of mature myeloid, erythroid and B-lymphoid cells after engraftment with normal h u m a n bone marrow or cord blood cells. Solid fragments of human fetal thymus, liver and bone marrow surgically implanted in SCID mice engraft, develop and can sustain long-term human haematopoiesis. Human blood-forming tissue grafts in SCID mice can also be seeded with selected precursor cell subsets, providing stem cell assays for thymus and bone marrow reconstitution {reviewed in ref. 85). These small animal models, in which human normal and diseased haematopoiesis can be conveniently recapitulated, appear particularly well suited for studying the multilineage dissemination of genes introduced into stem cells. As a proof of concept, 2050% CA18-resistant human CFU-GM were recovered from the marrow and spleen of bnx mice engrafted 4 months earlier with human marrow infected with a retrovirus carrying the neo gene. sz As previously discussed in

Gene Transfer into Stem Cells

detail, performing gene transduction in the presence of cultured haematopoietic stroma may permit the maintenance of long-lived progenitors. Nolta e t al. 6s have addressed this point by 'humanizing' bnx mice with bone marrow or GCSF-mobilized blood CD34+ cells submitted to retrovirus-mediated gene transfer in the presence or absence of adherent h u m a n irradiated stromal layers. Mice engrafted with cocultured progenitors contained significant numbers of h u m a n haematopoietic cells up to 11 months later, whereas CD34+ cells infected in the sole presence of recombinant retroviruses failed to establish sustained haematopoiesis. The SCID-hu chimera can also support the full sequence of h u m a n intrathymic lymphoid development from supplied haematopoietic stem cells while it has not yet been possible to model the entire T-cell lineage by in v i t r o culture (reviewed in ref. 86). SCIDhu mice are therefore of special interest to investigate foreign gene inclusion in h u m a n T-cell compartments via transgenic stem cells. Akkina et al. 44 transduced the neoR gene into human fetal liver CD34+ cells that were used to replete human fetal thymus grafts in SCID mice. At 4 weeks, 2% mature thymocytes in the grafts tested positive, by PCR, for neo integration. In an attempt to model the gene therapy of genetic or acquired diseases of the T-cell lineage diagnosed during pregnancy, cord blood CD34+ cells in which the mouse CD2-encoding gene had been transduced with high efficiency were similarly used to colonize SCID-hu thymuses. Five to 10 weeks later, mCD2 was detected by flow cytometry on up to 10% of human CD4+ thymocytes repopulating the grafts. Vector genomes were detected in graft cell DNA by Southern blotting, and analysis of integration sites permitted study of the contribution of distinct transgenic precursors to lymphopoiesis.4s Worth mentioning here are h u m a n sheep chimeras, in which human longterm haematopoiesis can be sustained

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from precursor cells infused i n u t e r o s9 and which could therefore be developed into powerful models of human transgenic haematopoiesis. Early successful experiments of gene transfer into mouse haematopoietic cells have also stimulated the development of blood cell gene therapy models-in monkeys, which would closely approximate human patients. Initially, transfer of autologous bone marrow cells infected with viral supernatants comprising ADA sequences into lethally irradiated rhesus monkeys led, in some instances, to short-term, low-level transgene expression in peripheral blood cells, s7 Cocultivation of monkey bone marrow with a packaging cell line expressing ADA in the presence of recombinant human IL-3 led to provirus detection in peripheral blood mononuclear cells--including granulocytes--up to one year p o s t transplant, s8 Since CD34 expression is also found in non-human primates in long-term reconstituting haematopoietic cells, the same group reproduced similar results by targeting purified C D 3 4 + C D l l b cells, a subset depleted of endogenous growth factor-producing cells instead of whole bone marrow. Multilineage haematopoietic reconstitution and ADA expression in T lymphocytes were documented. 88 Xu e t al. 6~ utilized M a c a c c a m u l a t t a sorted CD34+ cells to re-examine gene transduction using viral supernatants in the presence of IL3, IL-6 and SCF and reported persistence of the transferred human glucocerebrosidase gene in myeloid and lymphoid lineages, included in up to 14% of the B lymphocytes, more than a year p o s t engraftment. Therefore, preclinical studies in monkeys have been useful in evaluating clinically acceptable gene transfer protocols in large animals which are close to humans, and have stressed that low transgene expression is generally achieved in organisms of human size engrafted with gene-modifled haematopoietic cells compared with equivalent experiments performed in mice.

304 Transfus. Sci. Vol.18, No. 2 CLINICAL TRIALS Although many clinical trials are underway Isee a recent review in ref. 8}, we shall restrict ourselves to the description of the few published studies with sufficient follow-up to permit firm conclusions. The first encouraging conclusion is that good practice can ensure safe gene delivery to human beings: to date, there has been no report of detrimental consequences to patients of gene transfer treatments. Two types of trials have been undertaken: one where normal or neoplastic stem cells were transduced using marker genes and the other where the transferred genetic material was able to induce a biologic response of therapeutic value in the underlying disease. Trials using marker genes have been set up tO assess the efficiency and longevity of transduced cells administered to the patients. Cells might be normal haematopoietic precursors and the trial then gives insight, not only into the efficiency of transduction, but also into the kinetics of repopulation of the recipient by the stem cells. Cells might also be neoplastic and the results of the trial, depending on its design, might be of therapeutic import. In 1993, Brenner e t al. 9° published the results of their studies on transferring the neoR gene into haematopoietic marrow precursors from patients with acute myelogenous leukaemia and neuroblastoma. Marrows were collected when patients had achieved complete remission after intensive myeloablative therapy. An aliquot of the marrow was separated over ficoll-hypaque. Mononuclear cells were then incubated for 6 h with viral supematant before being cryopreserved. The efficiency of transduction, evaluated by numerating G 418-resistant CFU-GM, was between 4 and 6%. This aliquot was infused later at the time of transplantation, along with the remainder of the non-transduced marrow cells into patients conditioned with chemotherapy. Two patients with acute myelogenous leukaemia relapsed

67 and 180 days after transplantation. Some of the clonogenic blast cells expressed the neoR gene, indicating that relapse was at least partly due to cells harvested in apparent clinical remission. These data provide a rationale for e x v i v o bone marrow purging. In the other cases no relapse was observed, l° The presence of the neoR gene was assessed in marrow progenitors ICFU-GM and precursors of mixed colonies of granulocytes, erythroblasts, monocytes and megacaryocytes, CFUGEMM} and in peripheral myeloid, B and T cells. Three sets of data suggested that very primitive precursors had been transduced: I1} neoR + CFU-GM were detected as early as 1 month and as late as 3 years after transplantation; 121 neoR + CFU-GEMM were detected as early as 6 months and as late as 3 years; and 13} for up to 3 years neoR+ cells were detected among mature cells, neutrophils, T cells [including cytotoxic T-cell lines) and B cells. 91 These data fit with a model where committed progenitors are responsible for early engraftment as opposed to the more immature ceils lin this case CFU-GEMM) responsible for later grafting. In 1995, Dunbar e t al. 11 reported the results of-marker studies {using neoR vectorsl of CD34+ cells from marrow and peripheral blood of patients with multiple myeloma or breast cancer. Initially, CD34+ cells were collected from blood after treatment with cyclophosphamide and infusion of GCSF and, secondly, from bone marrow following administration of 5 fluorouracil. Aliquots from blood and marrow were then incubated with viral supernatant for 72 h in the presence of growth factors, IL-3, SCF and IL-6 for patients with breast carcinoma, and IL-3 and SCF for patients with multiple myeloma. Fresh viral supematant and g r o w t h factors were procured once

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daily. Two slightly different vectors, both containing the neoR gene, were used for either the bone marrow or the peripheral blood CD34+ cells. The efficiency of transduction, evaluated by counting the G 418-resistant CFU-GM, was approx. 20%. At the end of the transduction procedure samples were cryopreserved. Patients then received pretransplant conditioning therapy before being transplanted with transduced and non-transduced blood and marrow cells. After transplantation, samples from bone marrow and blood were collected for up to 24 months. All samples were positive {using semi-quantitative PCR) 15 days after grafting. In three patients {out of 10), samples were still positive 17 months after transplantation. Remarkably, some of the late positive samples showed signal provided by the vector used for transducing the peripheral blood CD34+ cells, which clearly indicated that these circulating cells had long-term reconstitutive ability. Although the estimated numbers of persistent transduced ceils appear to be one log higher in the study of Brenner e t al. 1° compared to that of Dunbar e t at., 11 both studies clearly indicate that it is possible to transduce cells with long-lasting reconstitutive ability, which are therefore close to, or identical to, self-renewing stem cells. Recently, engraftment of haematopoietic precursors transduced with a vector containing the human ADA gene has been described in neonates and children with SCID due to ADA deficiency. Kohn e t al. 12 collected CD34+ cells from the cord blood of three neonates with ADA deficiency. The retroviral vector comprised human ADA eDNA under the transcriptional control of LTR and the neoR gene under the control of an internal SV40 promoter. Fractions enriched in CD34+ cells were incubated for 3 days with viral supematants in the presence of IL-3, IL-6 and SCF. Each day fresh viral supernatant was added. The

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efficiency of transduction, evaluated by counting G 418-resistant CFU-GM, was 12, 20 and 21%. Transduced cells were injected intravenously into each patient on the fourth day of life. Samples of circulating granulocytes and mononuclear ceils were collected for up to 18 months following transplantation. Using PCR, with sense primer in the qt region of the vector backbone and antisense primer in the third exon of the human ADA eDNA, vector-containing cells were detected for the 18 months of observation, although at a very low frequency (1 3000 -1 to 1 100,000-t). At 1 year, marrow was collected and CFU-GM were examined for their resistance to G 418 indicating expression of the retroviral vector. The value {4-6%) was higher than that found in peripheral cells, suggesting better expression in immature cells compared to end cells. Using inverse PCR, as described by Nolta e t al. z2 the number of integration sites was measured and found to be at least 3-5. Eventually, the presence of ADA was checked, at the mRNA and protein levels, on cells amplified from G 418resistant CFU-GM. This trial showed that it was possible to transduce cord blood haematopoietic precursors from neonates with ADA deficiency using a vector containing the ADA gene. Longterm (at least 1 year) expression of the gene was found in progenitors and end ceils although at a very low frequency. This low frequency might be due to the lack of any myeloablative regimen before transplantation. In 1995 Bordignon e t al. 9 reported the results of their studies on transduction of bone marrow cells from two 2year-old children suffering from SCID. Retroviral vectors comprised the human ADA eDNA under the control of its own promoter {both inserted in the LTR region] and the neoR gene. T cell-depleted bone marrow cells were transduced by multiple exposures with viral supernatant in the presence of adherent layers of long-term marrow cultures and in the absence of growth factors, the culture time being 3 days. The efficiency of

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transduction, evaluated by numerating G 418-resistant CFU-GM, was approx. 40%. Peripheral blood T lymphocytes were also transduced using a slightly different vector. Transduced cells Iboth from marrow and peripheral bloodl were administered intravenously in several injections [5-9) over a period of 10-24 months. Monitoring of the T cells following transplantation revealed two waves of repopulation. For the first months after grafting most of the T cells showed signal provided by the vector used for transducing T cells. On the contrary, after one year, most T cells showed signal provided by the vector used for the bone marrow cells, indicating their marrow origin. These data therefore indicate the engraftment of transduced long-lasting precursors able to repopulate not only the myeloid compartment {as shown by the presence of G 418-resistant and ADA + CFU} but also the T-lymphoid compartment. The small number of marrow precursors giving rise to T cells suggested a selective advantage of transduced cells over non corrected ones, a hypothesis confirmed by the analysis of over 200 T-cell clones obtained at different times following grafting. These data, and that of Kohn e t al., 12 confirm that in humans it is possible to transduce multilineal long-lasting precursors from either bone marrow or cord blood. More recently, Hoogerbrugge e t al. 9z reported their experience with ADA gene transfer in CD34+ bone marrow cells from three children {1-5 years old} with ADA deficiency. CD34+ cells were cultured for 3 days on the irradiated packaging cell line delivering ADA-comprising vectors in the presence of 5 ng mL -1 of human IL-3. The efficiency of transduction, evaluated by counting XylA/dCF-resistant CFU-GM, was 5-12%. After transplantation, ADA eDNA was detected by PCR in circulating cells for no longer than 14 months and in marrow cells for no longer than 24 months. These data are somewhat at variance with those of Kohn e t al. 12 and Bordignon e t al. 9 Many differing

parameters may explain the difference in result, including the source of the transduced cells, the number of transplants and the means of transduction and retroviral constructs. Taken together, the clinical trials indicate that human haematopoietic precursors from variable sources Ibone marrow, mobilized peripheral blood or cord bloodJ can be transduced using vectors comprising either marker genes or genes of therapeutic interest. It is very probable, according to data from most of the studies, that a fraction of long-lasting, multilineal precursors responsible for late reconstitution are effectively transduced. The problem is the very low number of such transduced cells and a still lower number of end cells from their progeny. The reasons for such a low transduction rate may reside in unsatisfactory e x v i v o infection protocols due either to relatively inefficient vectors or to inadequate means of transduction, all problems that have been analyzed in this review. However, there remains one parameter independent of the e x v i v o protoc o l - t h e clinical status of the patients. The relevance of this parameter is suggested by the remarkably good results provided by Brenner e t al. 1° These investigators collected cells from young patients Imostly children) who had received heavy chemotherapy. In these patients the proportion of precursors in cycle may have been higher than in the patients studied by Dunbar e t a l . l l or patients with ADA deficiency. 9,12 The latter problem raises the question of conditioning the recipient either before collecting cells for gene transfer or at the time of transduced cell administration. Studies in dogs show that marrow ablation is not necessary for long-term maintenance of transduced cells 49,9a although myelosuppressive conditioning substantially improves engraftment. 94 In conclusion, the question at the present time is no longer whether human primitive haematopoietic precursors can be transduced, since the

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answer is yes. The key issue is that of the number of primitive cells in cycle at the time of transduction and effectively transduced at the time of transplantation. This number, difficult to evaluate, depends not only on " e x v i v o " parameters as examined in this review but also on " i n v i v o " parameters such as patient status, treatment regimen and administration of cytokines. Future studies will reveal how m a n y and how winding are the roads leading to this c o m m o n goal of infecting enough primitive cells to yield substantial therapeutic effect.

Acknowledgements We are grateful to Dr J.M. Heard for his review of this article, c o m m e n t s and suggestions.

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