Peripheral stem cells in bone marrow transplantation. Peripheral blood stem cell and gene therapy.

Mobilized peripheral blood stem cells characterized by sustained re-populating ability could be optimal target cells for ex-vivo gene transfer. In spite of very attractive preliminary results obtained in the murine studies, therapeutically efficient gene transfer and expression in human targeted cells must be proven. In recent years, effort has been spent on the identification of factors limiting gene transfer efficiency of haematopoietic stem cells. Increasing knowledge concerning haematopoiesis and gene transfer has helped in identifying a number of limiting factors. These factors as well as the strategies that showed increased retroviral infection of haematopoietic stem cells will be discussed. Finally, the results of the clinical trials will be reported.     

Onco-retroviral and lentiviral vector-based gene therapy for hemophilia: preclinical studies

Hemophilia A and B gene therapy requires long-term and stable expression of coagulation factor VIII (FVIII) or factor IX (FIX), respectively, and would need to compare favorably with protein replacement therapy. Onco-retroviral and lentiviral vectors are attractive vectors for gene therapy of hemophilia. These vectors have the potential for long-term expression because they integrate stably in the target cell genome. Whereas onco-retroviral vectors can only transduce dividing cells, lentiviral vectors can transduce a broad variety of cell types irrespective of cell division. Several preclinical and clinical studies have explored the use of onco-retroviral and, more recently, lentiviral vectors for gene therapy of hemophilia A or B. Both ex vivo and in vivo gene therapy approaches have been evaluated, resulting in therapeutic FVIII or FIX levels in preclinical animal models. Whereas in vivo gene therapy using onco-retroviral or lentiviral vectors often led to long-term FVIII or FIX expression from transduced hepatocytes, ex vivo approaches were generally hampered by either low or transient expression of FVIII or FIX levels in vivo and/or inefficient engraftment. Furthermore, immune responses against the transgene product remain a major issue that must be resolved before the full potential of these vectors eventually can be exploited clinically. Nevertheless, the continued progress in vector design combined with a better understanding of vector biology may ultimately yield more effective gene therapy approaches using these integrating vectors.     

The use of nonhuman primate models to improve gene transfer into haematopoietic stem cells

Primitive haematopoietic progenitor and stem cells (HSC) have been pursued as highly desirable targets for genetic therapy as technology allowing safe and controllable transfer of exogenous genes into eukaryotic cells was developed a decade ago. Retroviral vectors have been used for the majority of preclinical and clinical studies directed at these cells, because these vectors have a number of the necessary properties, including chromosomal integration, helper-free production systems, and lack of toxicity. Until recently, however, results with these vectors in clinical trials and large animal models indicated efficiency of gene transfer as a major hurdle to be overcome. We have focused on using the rhesus macaque autologous transplantation model to optimize gene transfer to primitive haematopoietic cells, and investigate questions regarding in vivo stem cell behaviour, in a system with proven predictive value for human haematopoiesis. By optimization of transduction conditions using standard vectors, gene transfer efficiency to primitive repopulating cells has reached the clinically relevant range of 5-20% long-term. Alternative vector systems, have also yielded promising results. We have also found that relatively simple manipulation of cell cycle status prior to reinfusion of marked cells results in significantly improved engraftment of transduced cells: this finding may have an impact particularly in the nonablative setting. The high level marking has permitted insertion site analysis and clonal tracking in vivo. Inverse PCR and/or a ligation-mediated PCR procedure have demonstrated that a large number of transduced clones (over 50) contribute to multiple lineages in vivo for up to at least 2 years post-transplantation. Thus far we have little evidence for rapid clonal succession or lineage-restricted engraftment of transduced cells. These and other advances should result in successful gene therapy for a variety of acquired and congenital disorders affecting HSCs and their progeny lineages.     

Ex vivo expansion of hematopoietic stem cells and gene therapy development

The results of a research project on ex vivo expansion of human haematopoietic stem cells (HSC) and development of gene therapy, funded by the Swiss National Research Program 46, are summarised and discussed in the context of current progresses and difficulties in these fields. A routine method for ex vivo expansion of human HSC is not yet available. However, stem cell biology has progressed importantly in recent years; ex vivo expansion of human HSC should become possible in the near future. Regarding gene therapy development, we obtained with HIV-1-derived bicistronic lentiviral vectors efficient delivery of genes into immature haematopoietic cells and also primary human B lymphocytes. However, clinical gene therapy still faces a variety of problems. For the (into chromosomes) integrating lentivectors, currently the most promising tools for HSC-based gene therapy, the risks of insertional mutagenesis need to be fully assessed before larger clinical trials can start.     

Lentiviral-mediated gene transfer into haematopoietic stem cells

OBJECTIVES: Lentiviral vectors can transduce nondividing cells. As most haematopoietic stem cells (HSCs) are nondividing in vivo, lentiviral vectors are promising viral vectors to transfer genes into HSCs. DESIGN AND SETTING: We have used HIV-1 based lentiviral vectors containing the green fluorescent protein (GFP) gene to transduce umbilical cord blood CD34+ and CD34+/CD38- cells prior to transplantation into NOD/SCID mice. RESULTS: High level engraftment of human cells was obtained and transgene expression was seen in both myeloid and lymphoid lineages. Bone marrow from the primary transplant recipients mice was transplanted into secondary recipients. GFP expression was seen in both lymphoid and myeloid cells in the secondary recipients 6 weeks posttransplantation. Human haematopoietic progenitor colonies were grown from both primary and secondary recipients. Over 50% of the haematopoietic colonies in these recipients were positive for the GFP transgene by PCR. Following inverse PCR, amplified fragments were sequenced and integration of the vector into human genomic DNA was demonstrated. Several vectors containing different internal promoters were tested in NOD/SCID mice that had been transplanted with transduced CD34+ and CD34+/CD38- cells. The elongation factor-1alpha (EF-1alpha) promoter gave the highest level of expression, both in the myeloid and lymphoid progeny of the engrafting cells. CONCLUSIONS: These data collectively indicate that candidate human HSCs can be efficiently transduced with lentiviral vectors and that the transgene is highly expressed in their progeny cells.     

Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.

The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.     

A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.     

Partial correction of murine beta-thalassemia with a gammaretrovirus vector for human gamma-globin

Several studies have demonstrated that recombinant lentivirus vectors containing extended globin gene expression cassettes and regulatory elements can ameliorate the pathogenic sequela in murine models of beta-thalassemia and sickle cell disease. Similarly promising results have not yet been obtained with recombinant gammaretrovirus vectors. Of these two vector classes, only gammaretroviruses have been tested extensively in clinical trials, with a proven ability to transduce long-term reconstituting hematopoietic stem cells with an exceedingly low incidence of serious side effects. Toward the continuing goal of developing retrovirus vectors for the treatment of the beta-chain hemoglobinopathies, we report here the assessment of a recombinant gammaretrovirus vector for human gamma-globin in murine models of beta-thalassemia. In the beta-thalassemia intermedia Hbbth-3/+ model, we observed a dose-dependent but transient increase in total hemoglobin and red blood cells, with a 2.5 +/- 0.2 g/dL increase in hemoglobin for transduction rates > or = 33%. In the severe beta-thalassemia major Hbbth-3/Hbbth-3 model, we observed a modest but statistically significant increase in survival, from a median of 15 days to 30 days (P = 0.001). These studies provide the first evidence that globin gene transfer vectors based on recombinant gammaretroviruses may provide a viable option for the treatment of the beta-chain hemoglobinopathies.     

In vivo silencing of the human gamma-globin gene in murine erythroid cells following retroviral transduction

Increased expression of fetal hemoglobin can ameliorate the clinical severity of sickle cell disease. Whereas temporary induction of fetal hemoglobin can be achieved by pharmacologic therapy, gene transfer resulting in high-level expression of the fetal gamma-globin gene may provide a permanent cure for sickle cell disease. We had previously developed a high-titer, genetically stable retroviral vector in which the human gamma-globin gene was linked to HS-40, the major regulatory element of the human alpha-globin gene cluster. Based on experience in transgenic mice, the truncated promoter of the gamma-globin gene of this vector should be active in adult erythroid cells. Our earlier studies demonstrated that this retroviral vector can give rise to high-level expression of the human gamma-globin gene in murine erythroleukemia (MEL) cells. We have now utilized this vector to transduce murine bone marrow cells that were transplanted into W/W(v) recipient mice. Analysis of transduction of murine BFU-e's in vitro and peripheral blood cells from transplanted mice in vivo demonstrated efficient transfer of the human gamma-globin gene. However, in contrast to the high level of expression of the human gamma-globin gene of this vector in MEL cells, the gene was completely silent in vivo in all transplanted mice. These observations confirm that all the necessary regulatory elements responsible for the developmental stage-specific expression of the human gamma-globin gene reside in its proximal sequences. They also emphasize the differences between gene regulation in MEL cells, transgenic mice, and retroviral gene transfer vectors. For this form of globin gene therapy to succeed, the proximal regulatory elements of the human gamma-globin gene may have to be replaced with different regulatory elements that allow the expression of the gamma-globin coding sequences in adult red cells in vivo.     

Specific transgene expression in human and mouse CD4+ cells using lentiviral vectors with regulatory sequences from the CD4 gene

Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4(+) cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from the CD4 gene--a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 10(7) infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2-deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4 gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4(+) T cells in reconstituted mice and in ex vivo-transduced human T cells. Non-T CD4(+) dendritic and macrophage cells derived from human CD34(+) cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4(+) T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4(+) cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.     

Gene therapy for the hemoglobin disorders

The hemoglobin disorders of beta-thalassemia and sickle cell disease together constitute the most prevalent group of human monogenic diseases. Although curative allogeneic stem cell transplantation therapy and palliative therapies have been developed for these disorders, the majority of patients still suffer significant morbidity and early mortality. The development of therapeutic approaches based on genetic manipulation of autologous stem cells therefore remains an attractive alternative. In the past 4 years, significant advances have been made toward this goal using lentiviral vectors to obtain high-level expression of complex globin gene cassettes. Therapeutic correction in murine models of both beta-thalassemia and sickle cell anemia has been achieved using this approach. These advances, coupled with progress in the ability to achieve in vivo selection of genetically modified cells, can now be evaluated in the well-developed nonhuman primate autologous transplant model. The goal in these studies is to provide preclinical safety and efficacy data prior to human clinical trials in order to maximize the likelihood of success in the context of an acceptable risk to benefit ratio. Here we review progress in each of these areas.     

Development of HIV vectors for anti-HIV gene therapy.

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.     

Gene therapy for the hemoglobin disorders

The hematologic disorders of beta-thalassemia and sickle cell disease are the most prevalent of human genetic diseases. Although palliative therapies and curative stem cell transplantation therapy have been developed for these disorders, treatment still remains suboptimal and many patients suffer significant morbidity and early mortality. Therefore, development of a gene therapy approach has been sought for many years. Major progress in the globin gene therapy field has been achieved by several laboratories. Using lentiviral vectors to obtain high-level expression of complex globin gene cassettes, therapeutic correction of several murine models of beta-thalassemia, and sickle cell disease was recently reported. This progress, coupled with developments in the ability to select and expand genetically modified stem cells in vivo, has advanced the possibility of gene therapy for the hemoglobin disorders in the near future. We review the developments in several areas that are critical for successful gene therapy of the hemoglobin disorders.     

Gene therapy of apolipoprotein E-deficient mice using a novel macrophage-specific retroviral vector

The use of retroviral gene transfer into hematopoietic stem cells for human gene therapy has been hampered by the absence of retroviral vectors that can generate long-lasting, lineage-specific gene expression. We developed self-inactivating retroviral vectors that incorporate gene-regulatory elements from the macrophage-restricted human CD68 gene. Through the transplantation of transduced murine hematopoietic stem cells (HSCs), we show that a vector incorporating a 342-base pair (bp) fragment of 5' flanking sequence from the CD68 gene, in addition to the CD68 first intron, was able to direct macrophage-specific expression of an enhanced green fluorescent protein (EGFP) reporter gene in inflammatory cell exudates and lymphoid organs in vivo. Levels of EGFP expression generated by this vector were greater than those generated by a standard Moloney murine leukemia retroviral vector, and they were stable for at least a year after transplantation of transduced HSCs. To evaluate the ability of this vector to generate therapeutically useful levels of gene expression, we transplanted apolipoprotein E (ApoE)-deficient HSCs transduced with a virus encoding ApoE into ApoE-deficient mice. Macrophages from these mice expressed levels of ApoE that were comparable to those from wild-type mice, and vector-driven expression of ApoE in macrophages was sufficient to reverse both hypercholesterolemia and atherosclerotic lesion development. The future application of this retroviral vector should provide a powerful tool to further elucidate macrophage function and for human gene therapy.     

The effect of multidrug-resistance 1 gene versus neo transduction on ex vivo and in vivo expansion of rhesus macaque hematopoietic repopulating cells

Transduction of murine stem cells with a multidrug-resistance 1 gene (MDR1) retrovirus results in dramatic ex vivo and in vivo expansion of repopulating cells accompanied by a myeloproliferative disorder. Given the use of MDR1-containing vectors in human trials, investigations have been extended to nonhuman primates. Peripheral blood stem cells from 2 rhesus monkeys were collected, CD34-enriched, split into 2 portions, and transduced with either MDR1 vectors or neo vectors and continued in culture for a total of 10 days before reinfusion. At engraftment, the copy number in granulocytes was extremely high from both MDR vectors and neo vectors, but the copy number fell to 0.01 to 0.05 for both. There were no perturbations of the leukocyte count or differential noted. After 3 cycles of stem cell factor/granulocyte colony-stimulating factor, there were no changes in the levels of MDR1 vector- or neo vector-containing cells. There was no evidence for expansion of MDR1 vector-transduced cells. Long-term engraftment with MDR1 vector- and neo vector-transduced cells occurred despite prolonged culture.     

In vitro toxicity of 3'-azido-3'-deoxythymidine, carbovir and 2',3'-didehydro-2',3'-dideoxythymidine to human and murine haematopoietic progenitor cells.

The myelotoxicities of three antiretroviral agents, 3'-azido-3'-deoxythymidine (AZT), carbovir (CBV) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), were evaluated in vitro with normal human and murine haematopoietic progenitor cells. These studies demonstrated that continuous AZT exposure was more inhibitory to human and murine colony formation than 1 h exposure, with murine and human progenitors similarly inhibited by continuous AZT exposure. These in vitro results on AZT's myelotoxicity correlate with both human and murine in vivo studies. CBV was only moderately toxic to human and murine cells following either 1 h or continuous exposure, with human and murine progenitors similarly suppressed by continuous CBV exposure. 1 h d4T exposure was less toxic to both human and murine marrow cells than continuous exposure and both species were equivalently inhibited when continuously exposed to d4T. In general, CBV was the least toxic agent to human and murine haematopoietic cells and AZT the most toxic. The study establishes CBV and d4T as less myelotoxic agents to human and murine haematopoietic progenitor cells in vitro than AZT which therefore could be considered as alternatives to AZT for the treatment of HIV infection.