Article

Gene Therapy of Hematopoietic Stem Cells: Strategies for Improvement

Published Online:https://doi.org/10.1152/nips.01343.2001

Abstract

Gene therapy of hematopoietic stem cells (HSC) is limited by low frequency of the target cells, their quiescent nature, poor engraftment of treated HSC, and lack of a selective growth advantage of genetically modified cells. Lentiviral vectors combined with positive selection strategies using conditional cell-growth switches should allow for improvement.

The genetic modification of hematopoietic stem cells (HSC) offers an enormous potential for the treatment of genetic diseases of hemopoiesis and lymphopoiesis, because of the ability to provide permanent correction. However, the ability to reach and to maintain therapeutically relevant fractions of genetically modified stem cells and their progeny after gene delivery presents major challenges to current HSC gene therapy. This is because of the low frequency of the target cells, the most likely quiescent nature of the most primitive HSC, unfavorable effects of in vitro cell culture on the engraftment potential of HSC, and, ultimately, the lack of a selective growth advantage of the genetically modified cells after engraftment for most diseases. In this review, we outline existing strategies and more recent approaches to improve engraftment of genetically modified stem or progenitor cells and also to confer selective growth advantage to these cells.

HSC as targets for gene therapy

Self renewal and differentiation.

One main reason for the choice of HSC as targets for gene therapy is their lifelong ability to self renew and to differentiate into specific cell types (Fig. 1). HSC are not fully characterized, but by experimental evidence a pool of cells expressing the surface glycoprotein CD34 and lacking CD38 (CD34+/CD38) was shown to contain cells with the highest potential for long-term reconstitution of the hematopoietic system. It was also demonstrated that repopulating HSC undergo multiple divisions in cell culture in serum-free media containing multiple growth factors and cytokines, such as stem cell factor (SCF), fms-like tyrosine kinase-3 (Flt-3) ligand, thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF), and interleukin (IL)-6 and -3 (7). A portion of these stimulated cells retain an engraftment potential as defined by multilineage engraftment in a surrogate nonobese diabetic (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay in mice.

           FIGURE 1.

FIGURE 1. Functional hematopoietic compartments. The self-renewal capacity decreases with progressing cell differentiation, whereas proliferation of cells increases dramatically along terminal differentiation in hemopoesis from progenitor to effector cells. LTCIC, long-term culture-initiating cells; CFU-S, colony-forming units-spleen; CFU-GEMM, common precursor colony-forming units for granulocytes, erythrocytes, monocytes, and megakaryocytes; G, granulocytes; M, monocytes; Eo, eosinophils; b, basophils; Meg, megakaryocytes; BFU-E, burst-forming units-erythroid; B, B lymphocytes; T, T lymphocytes; Neutro, neutrophils; Mac, macrophages; Tc, thrombocytes; Ec, erythrocytes.


Mobilization.

The frequency of circulating CD34+ cells in the peripheral blood is ~0.1% but can be increased to 1% by the use of growth factors, e.g., G-CSF. Uchida et al. (19) showed that, after in vivo stimulation of HSC with G-CSF and release into peripheral blood, these cells remain in the G0/G1 cell cycle phase, whereas stem cells directly harvested from bone marrow are actively cycling. Compared with marrow cells, HSC mobilized with G-CSF show a 24-h delayed entry into cell cycle after in vitro culture and stimulation with cytokines. Stem cell quiescence is important for preservation of the stem cell compartment and protection from myelotoxic injury. It is, on the other hand, a major obstacle for retroviral gene transfer into HSC. Because the retroviral vector needs breakdown of the nuclear membrane during mitosis for integrating the vector DNA into the host genome, it cannot transduce quiescent, nondividing cells.

Homing and engraftment.

Homing is the first step in the engraftment process. After bone marrow transplantation, migration of the transplanted cell pool from the peripheral blood into the recipient's bone marrow or functional equivalent is necessary (homing) to reconstitute the hematopoietic system (engraftment). The basis for HSC homing is the interaction of selectins, integrins, and the immunoglobulin family that mediate cell-to-matrix and cell-to cell interactions (15). Recently, it has been observed that the chemokine stromal cell-derived factor 1 (SDF-1) and its receptor CXC-motif chemokine receptor 4 (CXCR4) are involved in engraftment of human repopulating stem cells into murine bone marrow. CXCR4 is expressed on CD34+ cells, among others, and antibodies against CXCR4 prevent engraftment (13).

Characteristics of oncoretroviral vectors.

Retroviral vectors are the main vehicles used for gene transfer into HSC. During the natural life cycle of a retrovirus, viral DNA is integrated into the chromosomal DNA of the infected target cell. This integration is random but stable. After cell division, the integrated DNA is passed on to the daughter cells and is expressed in them. Vectors used in present clinical gene therapy trials are based on the Moloney murine leukemia virus (MMLV), an oncogenic retrovirus. Genetic engineering for vector development retains the capacity of DNA integration into the host genome but eliminates the vector's ability to produce replication-competent retroviruses. Such modified vector viruses lack the sequences coding for the essential viral proteins, the group-specific antigen (Gag), reverse transcriptase (Pol), and envelope (Env). Packaging of the viral RNA into viral particles must be performed in so-called packaging cell lines, which express and provide the viral proteins Gag, Pol, and Env in trans (Fig. 2).

           FIGURE 2.

FIGURE 2. Technique of oncoretroviral gene addition [for X-linked chronic granulomatous disease (CGD)]. The therapeutic transgene to be transferred (e.g., gp91phox for X-CGD), together with a basic retroviral backbone containing just the long terminal repeat (LTR) promoter and enhancer regions and the packaging signal (Ψ+) is introduced into the packaging cells by plasmid transfection. The packaging cell line is constructed such that the group-specific antigen (Gag), the envelope (Env), and the polymerase and reverse transcriptase (Pol) are constitutively expressed and therefore provided in trans. These trans-acting factors are needed for packaging of the expressed vector RNA into retroviral particles. On infection of a target cell, the transferred vector RNA is reverse transcribed by Pol (reverse transcriptase) into double-stranded DNA for random integration into the cell's genome and subsequent expression of the transgene.


Limitations of current gene therapy into HSC

Low frequency of the target cell.

The long-term repopulating stem cell seems to be located within the CD34+/CD38 fraction of the bone marrow. The marrow contains ~1–2% CD34+ cells, of which only 1% are CD34+/CD38. Thus only 1 in 106 bone marrow cells is the desired target cell for gene therapy. It is possible to harvest a portion of these cells from the marrow itself by multiple needle aspirations or after G-CSF-induced mobilization from the peripheral blood by a leukapheresis machine. After mobilization, the level of CD34+ cells in the peripheral blood does not rise beyond 1% of all nucleated peripheral blood cells again.

For an autologous stem cell transplantation, >2 × 106 CD34+ cells (10 ml marrow)/kg recipient body wt or 5 × 104 CD34+/CD38 cells/kg are needed. This cell harvest can just be reached for clinical purposes, but it is technically impossible to extract 10% or more of the body's total HSC, which is optimal for ex vivo gene transfer.

Quiescent nature of the target cell.

The CD34+/CD38 cells are very small and are in G0 phase, with densely packed DNA and intact nuclear membrane. It is not possible to transfect these cells by oncoretroviral gene transfer, because this requires breakdown of the nuclear membrane during mitosis for integrating the vector DNA into the host genome. Transduction is only possible after in vitro culture of HSC with cytokines that enable both induction of cell division and retention of repopulating capacity.

Poor engraftment after cell activation.

Exposure of HSC to cytokines (murine IL-3 and -6, rat SCF) for oncoretroviral transduction results in a decrease of engraftment of these cells when competed with unmanipulated HSC. When the two populations of cells were administered simultaneously into marrow-ablated isogenic mice, the extent of engraftment of cytokine-treated cells depended on the numbers of injected fresh cells. The more unmanipulated cells injected, the less engraftment of manipulated cells occurred. On the other hand, when cytokine-treated cells were given 2 or 4 days before unmanipulated cells, there was a much greater engraftment of these cells (14). These competition experiments have clear implications for clinical gene therapy protocols.

The cytokine-induced defect of engraftment is cell cycle dependent. Habibian et al. (9) showed in 1998 that the repopulation ability of the HSC fluctuates with cell cycle transit. After taking mice HSC into culture, the first cycle transit from dormancy through mitosis took ~36–40 h. The engraftment of marrow cells exposed to SCF and IL-3/-6/-11 was assessed at 2- to 4-h intervals of culture by using irradiated hosts. Remarkably, no engraftment occurred in late S and early G2 phase. The repopulation defect was reversible after mitosis and resumed with the next cell cycle.

Data showing that there are dramatically different outcomes with stem cell engraftment based on the precise time and cell cycle at which cells are injected intravenously into irradiated mice now also exist for human HSC (8). Human HSC were cultured in the presence of SCF, Flt-3 ligand, and IL-3/IL-6/G-CSF. Cultured cells were sorted into G0/G1 and S/G2/M CD34+ cells. On the whole, engraftment was only seen in NOD/SCID mice transplanted with G0/G1 cells but not with cells in S/G2/M phase. These data are bad news for the clinical utility of current oncoretroviral transduction protocols, since they depend on maximum induction of stem cell cycling. The basic factors underlying the silencing of the engraftment potential of stem cells still have to be analyzed.

Lack of selective growth advantage postengraftment.

In a very few diseases, there is a natural selective advantage of gene-transduced cells, so that pretreatment of the recipient by cytotoxic agents or other means is not necessary for long-term engraftment. Fischer's group (5) reported in 2000 the successful gene therapy of SCID-X1 disease in two patients. SCID-X1 is an X-linked inherited disorder characterized by defective T cell and natural killer (NK) cell development, leading to the absence of T and NK cells. The underlying causes of the disease are genetic defects in the common γc cytokine receptor subunit that forms an essential part of multiple cytokine receptors (e.g., of IL-7 and -15 cytokine receptors transmitting survival and proliferative signals to T and NK progenitors). The two study patients received no cytotoxic conditioning before infusion of CD34+ cells transduced with an oncoretroviral vector expressing the γc transgene. T lymphocytes became detectable in both reported patients at days 30 and 60, respectively, and the expression of NK cells could be observed at days 30 and 150, respectively. In both patients, γc expression of T and NK cells persisted up to at least 10 mo. Oncoretroviral gene therapy of SCID-X1 disease was thus able to completely restore the cellular immunity against pathogens. Immune reconstitution was even more rapid than after allogeneic bone marrow transplantation, so that the two children could leave the hospital 1–2 mo earlier than expected. The success of this clinical trial can be attributed to the fact that modified cells carrying the γc transgene had a strong selective growth advantage, because there are no competing defective T and NK cells in this special disease.

In most other genetic diseases of the bone marrow, there is an unfortunate competition between transduced and nontransduced cells. In another clinical trial in 2000, Choi and Malech (6) did show that competition was evident during ex vivo gene therapy for chronic granulomatous disease (CGD). Again, this trial was carried out without conditioning the patients' marrows with cytotoxic drugs. Purified CD34+ peripheral blood cells were transduced with an oncoretroviral gp91phox vector and were reinfused in repetitive runs 50 days apart. Very few corrected (NADPH oxidase-positive) cells could be detected in the marrow in four of the six treated patients at 6–8 wk after infusion, peaking at only 1 in 500 to 1 in 3,000 corrected cells after each cycle of gene therapy. Clearly, in CGD the corrected phagocytes do not have a natural selective growth advantage over the majority of defective cells, so that introduction of an artificial selection advantage will be needed in the future to change the clinical phenotype (a minimum of 5–10% transduced cells will be required).

Strategies to circumvent limitations

Improving engraftment

purification of transduced cells from competitors.

This strategy makes use of a cell surface marker gene, which is only expressed on successfully transduced cells and allows for positive selection and enrichment of transduced cells before reinfusion into the patient. Although this regimen does not provide an in vivo selective growth advantage to transduced cells, it may help to avoid the competition of transduced and nontransduced cells when engrafting into the hematopoietic niches. The gene for the low-affinity nerve growth factor receptor (LNGFR) in a truncated form has been proposed for this purpose (17). Since it is a human gene, no immune response is expected. By using anti-LNGFR antibodies, sorting of transduced cells is possible. We, in collaboration with others (Sadat MA, Pech N, Saulnier S, LeRoy B, Hossle JP, Grez M, and Dinauer MC, unpublished observations) have shown in a mouse model that sorting of LNGFR-positive cells before marrow transplantation improves the levels of reconstitution and gene expression.

In humans, this approach has serious limitations, since the frequency of transduced cells is very low and elimination of all nontransduced cells would probably result in a graft unable to support a normal hematopoiesis. Administering the nontransduced cells several weeks after the sorted transduced cell would be feasible but would result in a prolonged aplasia period if previous chemotherapy had been used.

gene transfer into “quiescent” hsc by lentiviral vectors.

Lentiviruses (such as HIV type I) encode proteins that permit transport of the retrotranscribed viral genome into the nuclei of nondividing cells through an intact nuclear membrane. Use of lentiviral vectors should therefore enable transduction of quiescent HSC and thus enable avoidance of the engraftment defect seen with HSC manipulated for uptake of oncogenic retroviruses. In contrast, vectors based on oncogenic retroviruses must wait for the nuclear membrane to break down during cell division before they are able to access the host DNA.

Complete reverse transcription of a lentiviral vector needs progression to the G1b phase of the cell cycle (11). The G1b phase is defined as the stage at which RNA levels are equivalent to those seen in early S phase before DNA synthesis. It has been suggested that it is the low levels of deoxynucleotide triphosphates in G0 phase that require transition to G1b. Further progression within the cell cycle to the S/G2/M phase is not required for successful lentiviral transduction of nucleated cells but increases the transduction efficiency.

Lentivirally transduced human HSC are clearly able to repopulate the NOD/SCID model mouse. This was shown by transduction experiments carried out on human umbilical cord blood HSC in the absence of cytokine stimulation (12). Expression of the green fluorescence protein (GFP) gene was detected up to 22 wk after engraftment (peak expression after 15 wk), and differentiating human cells were found to repopulate the mouse bone marrow. However, no transduced cells were seen when using oncoretroviral vectors. There is a general consensus that lentiviral vectors need minimum cytokine stimulation, which should not endanger marrow-reconstituting potential. In 2000, Brenner et al. (3) proposed a multicolor cotransduction method as a convenient tool to define optimum transduction conditions for lentivectors while minimizing the cell cycling required for oncoretroviral vectors. They introduced a cyan fluorescence protein (CFP) gene into a self-inactivating lentivector and a GFP gene into an oncoretroviral vector. A low cytokine dose of SCF (10 ng/ml), Flt-3 ligand (50 ng/ml), and TPO (10 ng/ml) allowed CFP lentivirus transduction of G0/G1 cells but no transduction of the same cells with GFP oncoretroviruses. Increasing the concentrations and number of growth factors augmented both lentivector and oncoretroviral vector transductions. The reconstitution potential of the HSC cotransduced in vitro by the two different vectors will have to be tested in NOD/SCID mice as an in vivo transplantation model.

A not-yet-completely resolved question is the provision of stable packaging cells for lentivector production. There has been difficulty in generating these cell lines because of the cytotoxicity of some of the HIV proteins and of the G glycoprotein of the vesicular stomatitis virus envelope, but recent progress is encouraging.

Conferring selective growth advantage

nonselective killing of competitors before transplantation by myelosuppressive conditioning.

Traditionally, it has been considered that space needs to be cleared in the HSC niches by myeloablation for adequate HSC engraftment. Recent observations suggest that engraftment is dependent on the ratio of host to donor HSC but not on any actions in clearing space. If this is in fact the case, treatments, which could selectively diminish HSC number/function without major myelotoxicity, should be able to markedly increase the percentage of donor chimerism by increasing the competitive advantage of transduced stem cells. The level of engraftment is cell dose related, so that higher numbers of HSC can overcome engraftment resistance. In humans and large animals, graft sizes necessary for reconstitution of a nonablated recipient are unfortunately too large to be provided without harm to the donor. Therefore, some degree of myelosuppressive conditioning has to be used in the recipient.

In 1995, Barquinero et al. (1) showed that acceptable degrees of autologous engraftment can be achieved in the dog model either by sublethal cyclophosphamide conditioning or sublethal total body irradiation (at 200 or 300 cGy). Four weeks after transplantation of gene-transduced cells, the levels of engraftment were 18% with sublethal cyclophosphamide and 33% with sublethal total body irradiation. Most remarkably, this level of autologous chimerism was observed up to 12 mo after transplant. In 1999, Rosenzweig et al. (16) showed durable engraftment in nonhuman primates (rhesus monkeys) after nonmyeloablative conditioning with 320–400 cGy. A high level of gene marking (10–15%) persisted for 4 mo.

In humans, cyclophosphamide conditioning has been used for aplastic anemia for many years, whereas sublethal irradiation with 200 cGy has only come into clinical use recently as the so-called Seattle protocol for allogeneic minitransplantation. Both maneuvers are indeed well tolerated in both the short and long terms, so that even repeated conditioning with these protocols would be possible should the effect of gene transduction wane with time.

selective killing of competitors after engraftment by cytotoxic drugs with shielding of transduced cells by resistance genes.

One attractive way to confer a selective growth advantage onto gene-transduced cells is the cotransduction of another gene coding for chemoresistance. The multidrug resistance gene 1 (MDR1) encodes a 120-kDa transmembrane protein (P-glycoprotein). It confers resistance to a variety of chemotherapeutic agents, including anthracyclines, vinca alkaloids, etoposide, and taxol. P-glycoprotein is an ATP-dependent cellular efflux pump for many lipophilic compounds. By cotransfecting the therapeutic transgene together with the MDR1 gene, protection of the gene-transduced cells against chemotherapy-induced myeloablation can be achieved.

Although attractive in theory, there are significant drawbacks to this approach. First, there is no true expansion of the desired transduced HSC, only an elimination of the nontransduced cells. At present, when only a few percent of the HSC can be harvested and transduced, elimination of >95% of nontransduced cells would probably result in aplastic anemia. In addition, the host would be exposed to recurrent cycles of chemotherapy with side effects on nonhematopoietic cells. Finally, a high peripheral white blood cell count has been seen in mice transplanted with expanded MDR1-transduced stem cells (4). Although this may be an unfortunate event caused by insertional mutagenesis, it demonstrates the risks of such a procedure.

In summary, the selective elimination of nontransduced cells after engraftment by chemotherapy, sparing protected cells, does not seem to be a promising avenue.

selective expansion of transduced cells by modified growth factor receptors.

Instead of relying for in vivo selection on genes that confer resistance to cytotoxic drugs with all inherent risks, it is also possible to use a cell growth switch allowing a minor population of transduced cells to be amplified ex vivo or in vivo (10). For this purpose, fusion proteins composed of growth factor receptor signaling domains are linked to one or more binding sites for a chemical inducer of dimerization. By administering the dimerizer, a small synthetic molecule, therapeutically relevant expansion of the wanted cells can be achieved, even if selection is restricted to the transduced HSC only (Fig. 3). The dimerizer could be given repeatedly over time, when needed. Biological responses would be kept within the desired range by titrating the dose of the dimerizer.

           FIGURE 3.

FIGURE 3. Selective expansion of transduced cells. Corrected cells, transduced with the therapeutic transgene, would be cotransduced with a hormone-inducible chimeric receptor, a fusion protein composed of a truncated granulocyte-colony stimulating factor (G-CSF) receptor [lacking the cytokine binding site, but still containing the transmembrane domain (TM) and the signaling domain (SIG)], and a mutated form of the hormone-binding domain of the estrogen receptor (ER), specifically binding to 4-hydroxytamoxifen. By administering tamoxifen, specific receptor dimerization and subsequently signaling would be induced. This would lead to selective cell division, e.g., expansion of functionally reconstituted, e.g., superoxide anion (O2)-producing, cells.


Several systems for expansion of gene-transduced HSC have been proposed. Only one, which seems suitable for in vivo application, will be described here. It consists of a truncated G-CSF receptor (lacking the cytokine binding site but preserving the cytoplasmic signaling domain) and a mutated form of the hormone-binding domain of the estrogen receptor. This mutated receptor no longer reacts with estrogen but binds specifically to 4-hydroxytamoxifen (20). The pharmacological property of tamoxifen (precursor of 4-hydroxytamoxifen) is well characterized, and its safety has been established through clinical use. Although promising in principle, we still lack animal experiments showing clinical benefit of the induction of cell division and demonstrating that transduced HSC are not exhausted by the maneuver.

Approved CGD gene therapy protocol (1st generation)

Our group at the University Children's Hospital in Zurich (in close collaboration with the groups of M. Grez in Frankfurt aum Main and D. Trono in Geneva) tries to correct the NADPH oxidase deficiency of phagocytes in the X-linked form of CGD (gp91phox deficiency). This bone marrow disease has the advantage that 5–10% of gene-corrected cells are sufficient for a healthy phenotype and the disadvantage of no selective growth advantage of gene-corrected cells. Our groups have previously demonstrated the correction of gp91phox deficiency either by an oncoretroviral vector (2) or a lentiviral vector (18). Knowing the limitations of gene transfer into HSC, two protocols have been or are being developed, a first-generation protocol clinically active in Germany since January 2001 and a second-generation protocol presently being tested (Fig. 4). Both protocols aim at achieving a clinically significant benefit (resolution of preexisting and freedom from new infections/granulomatous inflammation) as expected after a significant correction of the disease (achievement of 5–10% gene-corrected cells over 3–6 mo).

           FIGURE 4.

FIGURE 4. Actual and future retroviral transduction protocols. SC, stem cells; Prom, tissue-specific promoter; SD, splice donor site ; SA, splice acceptor site; ires, internal ribosomal entry site; BD, hormone-binding domain.


In the first-generation protocol, an oncoretroviral vector is being used containing the gp91phox transgene under the regulation of the oncoretroviral long-terminal repeat. The transduction protocol uses a closed bag system, fibronectin to approximate viruses and cells, and four cytokines: SCF, Flt-3 ligand, IL-6, and TPO. IL-3, which strongly stimulates cell division and is associated with poor engraftment of activated cells (3), was omitted. In large-scale transductions, 50–70% of CD34+ cells can be transduced. We demonstrated the repopulating capacity of transduced cells after cytokine activation by NOD/SCID experiments revealing normal engraftment, proliferation, and differentiation of the human HSC. Up to 60% of the granulocyte/macrophage colonies demonstrate expression of the transgene gp91phox.

After stem cell mobilization into peripheral blood, HSC are harvested, transduced, and, later, reinfused. The CGD patient is prepared by myelosuppressive conditioning with 4 × 50 mg/kg of cyclophosphamide. Ethical approval for this conditioning has been obtained by the expert committee for somatic gene therapy of the Bundesärztekammer Deutschland. We hope that we will achieve long-term engraftment in the patient of 5–10% of gene-transduced cells. Gene expression for 3–6 mo duration would be beneficial to a CGD patient, since this clears subclinical infections (eliminates occult infectious agents within granulomas) and improves organ function (e.g., lung function after resolution of granulomas).

Planned CGD therapy protocol (2nd generation)

In the future, we are planning to replace the oncoretroviral vector with a lentiviral vector, which for transduction only needs an exposure of ~12 h with TPO (but no other cytokine) to get into G1b phase. The gene-transduced HSC would then be expected to have a normal engraftment potential. They would be reinfused into a CGD patient without previous cytotoxic conditioning. They would carry a second transgene containing a cell growth switch, a fusion gene with a hormone-binding domain, and a truncated G-CSF receptor. Administration of the dimerizer tamoxifen ex vivo and in vivo when needed would be expected to result in stem cell expansion overgrowing the nontransduced cells. Since the lentiviral vector would integrate into noncycling stem cells, it would be expected to provide long-term, if not permanent, correction of the disease.

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