Retrovirus-mediated gene transfer into human hematopoietic stem cells

 Human hematopoietic stem cells genetically modified by retroviral-mediated gene transfer may offer new treatment options for patients with genetic disease. The potential of gene-modified hematopoietic stem cells as vehicles for gene delivery was first illustrated by the demonstration that hematopoietic systems of lethally irradiated mice can be reconstituted with retroviral vector transduced syngeneic bone marrow, and that these cells can in turn provide genetically marked progeny which persist in blood and marrow over extended time periods [1–4]. In contrast, hematopoietic stem cells from large animals prove difficult to transduce with retroviral vectors and are consequently less likely to function as vehicles for long-term gene therapy. Indeed, clinically relevant levels of gene transfer into large animal and human hematopoietic stem cells has not been widely achieved. The need for improved retroviral vector systems and for understanding the biology of hematopoietic stem cell gene transfer continue to fuel intense research activity. Preliminary results from human stem cell gene marking and gene therapy trials currently underway are encouraging. This contribution reviews the underlying concepts relevant to retroviral-mediated gene transfer into hematopoietic stem cells. We survey the evolution of approaches for gene transfer into hematopoietic stem cells, from murine and large animal models to the first human clinical trials. Finally, we discuss new strategies which are currently being pursued. Received: 12 March 1997 / Accepted: 21 July 1997     

Towards liver-directed gene therapy: Retrovirus-mediated gene transfer into human hepatocytes

Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either theLacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins,E. coli -galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans.     

Cholinergic neural transplants into hippocampus restore learning ability in monkeys with fornix transections

Summary Monkeys with bilateral transections of the fornix were severely but selectively impaired at learning visuospatial conditional tasks presented in a Wisconsin General Test Apparatus. Bilateral transplantation of cholinergic-rich embryonic basal forebrain tissue into the hippocampus led to complete recovery from this specific learning impairment across a range of task difficulties. Administration of the direct cholinergic agonist pilocarpine to ungrafted animals immediately before testing also reduced this impairment, suggesting that the graft-associated recovery was mediated by acetylcholine release. Transection of the fornix produced a marked loss of acetylcholinesterase (AChE) staining confined to hippocampus and entorhinal cortex relative to controls. In all transplanted animals densely AChE-staining cellular masses were seen bilaterally in temporal lobe structures, with fibre outgrowth into surrounding host tissue.     

In vivo magnetic resonance spectroscopy of human fetal neural transplants.

To better define the survival and cellular composition of human fetal neurotransplants in vivo, we performed quantitative 1H MRS to determine the concentration of the neuronal amino acid [N-acetylaspartate] within MRI-visible grafts. In all, 71 grafts in 38 patients [24 Parkinson's disease (PD), 14 Huntington's disease (HD)] were examined, as well as 24 untreated PD and HD patients and 13 age-matched normal controls. MRI appearances of edema were present in three out of 71 grafts, the remainder being consistent with histologically identified viable neural transplant tissue. N-acetylaspartate (NAA), creatine, choline, myoinositol and glutamine plus glutamate (Glx) were identified in all post-transplant putamens, with abnormal metabolites, lactate and/or lipid detectable in only three patients. Of 71 grafts, 19 occupied more than 60% of the MRS-examined volume (VOI) (mean 84.2 +/- 3%; range 61-100%). In those, [NAA] was 8.50 +/- 0.99 mM in eight PD spectra and 6.59 +/- 0.81 mM in 11 HD spectra, and was not significantly different from controls. In contrast, transplanted fetal neurones contain less than 0.4 mM of the neuronal amino acid NAA. This suggests that established fetal neurotransplants in the human putamen of both PD and HD patients are populated by adult neurones, axons and dendrites.     

Analysis of adenovirus gene transfer into adult neural stem cells

Adult neural stem cells (aNSCs) represent an attractive source for the production of specific types of neurons in degenerative CNS diseases and for the development of new regenerative gene therapies. However, the use of adult NSCs for transplantation and gene replacement strategies requires efficient gene expression in the cells. Due to the low pathogenicity of adenovirus (Ad) for humans, its large delivery capacity, and long-term transgene expression, Ad vectors are widely used. Here, we tested the potential of the Ad vector system to transduce adult NSCs. Analysis of Ad receptor expression in primary aNSCs revealed a complete lack of the coxsackie-adenovirus receptor and no or low expression of alphanu- and beta5-integrins, respectively, on mRNA and protein level. Consistently, transduction at different multiplicities of infection using an Ad vector expressing the enhanced green fluorescent protein (GFP) showed that adult NSCs are particularly resistant to Ad infection even at highest MOI (1000) in contrast to differentiated types of neural cells.     

Retrovirus-mediated gene transfer of receptor activator of nuclear factor-kappaB-Fc prevents bone loss in ovariectomized mice

Postmenopausal osteoporosis is characterized by increased bone resorption due to estrogen deficiency. Receptor activator of nuclear factor-kappaB-Fc (RANK-Fc), a fusion protein that specifically blocks receptor activator of nuclear factor ligand binding to RANK, has been known to be efficient and well tolerated in animal models of osteoporosis. Here we show that cell-based gene therapy with RANK-Fc effectively prevented bone loss in ovariectomized (OVX) mice. Thirty-one young adult female C57Bl/6 mice were used, and repeated intraperitoneal injection of mesenchymal stem cells (MSCs) transduced with retrovirus was performed as follows: 1) Sham-operated mice (n = 8); 2) OVX mice treated with phosphate-buffered saline (OVX-PBS; n = 8); 3) OVX mice injected with MSCs transduced with control retrovirus (OVX-green fluorescent protein [GFP]; n = 7); and 4) OVX mice injected with MSCs transduced with RANK-Fc (OVX-RANK-Fc; n = 8). Cellular expression of RANK-Fc was confirmed by Western blot analysis of cell lysates and conditioned medium and also by enzyme-linked immunosorbent assay for the mice serum. Measurement of bone mineral density (BMD) by dual-energy x-ray absorptiometry (PIXImus) revealed that the OVX-RANK-Fc group gained significantly higher BMD than either the OVX-PBS group or OVX-GFP group after 8 weeks. The expression of GFP, which is coexpressed with RANK-Fc, was detected by polymerase chain reaction analysis of DNA isolated from femur and intra-abdominal fat, whereas no GFP signal was identified in liver, brain, heart, lung, or bone marrow aspirates. These suggest that expression of RANK-Fc by genetically modified MSCs may be a feasible option for the prevention of bone loss induced by ovariectomy.     

Differentiation and histological analysis of embryonic stem cell-derived neural transplants in mice

We report here that neural transplantation of in vitro-differentiated embryonic stem (ES) cells provides a versatile strategy for gene transfer into the central nervous system. ES cells were subjected to an optimized in vitro differentiation protocol to obtain embryoid bodies. These aggregates were stereotaxically transplanted into the brain of recipient adult mice, where they followed a strictly controlled differentiation pattern and eventually formed mature neural grafts. A marker gene, introduced into the ROSA26 locus allowed for precise determination of the fate of the descendants of the transplanted embryoid bodies and revealed that not only neurons but also astrocytes, oligodendrocytes and even microglial cells were graft-derived. Evaluation of long-term experiments showed viable grafts with a stable transgene expression and proved that this approach provides a tool for reliable gene expression within a spatially delimited area of neural tissue.     

Retinal transplants into the anterior chamber of the rat eye

Developing retinas from 13-18-day fetuses and 2-day neonatal Long-Evans rats transplanted into the anterior chamber of adult eyes of the same or different strain (Lewis) survive and differentiate. Light and electron microscopic studies show that the transplants undergo histogenetic differentiation, resulting in the development of neurons and Müller glial cells and formation of nuclear and plexiform layers. Vascular connections develop between the host iris and the retinal transplant. Vessels and nerves, presumably of iridal origin, were seen on the surface of some transplants. Possible manifestations of graft rejection were monitored; signs of tissue rejection in transplants performed in the Long-Evans rats, an outbred strain, were rare and if present they were mild, at least during the survival periods of up to 91 days allowed in these experiments. Transplants into the eyes of Lewis rats were also well tolerated during the survival period. These observations indicate that retinal transplantation to the adult eye of a genetically different host can be successfully achieved and that both embryonic and perinatal retinas are suitable as donor tissue for ocular transplants. The procedure offers ample opportunities for the study of problems related to retinal plasticity.     

Effects of cholinergic-rich neural grafts on radial maze performance of rats after excitotoxic lesions of the forebrain cholinergic projection system--I. Amelioration of cognitive deficits by transplants into cortex and hippocampus but not into basal forebrain.

After ibotenate (10.0 mg/ml) lesions to the nucleus basalis and medial septal regions, at the source of the cortical and hippocampal branches of the forebrain cholinergic projection system, rats displayed long-lasting stable impairment in reference and working memory in both spatial (place) and associative (cue) radial maze tasks. Cell suspension transplants of cholinergic-rich fetal basal forebrain tissue dissected at embryonic day 15 substantially improved all aspects of radial maze performance to a comparable degree whether sited in cortex, hippocampus, or both regions of the host brain. No additive effects were obtained with grafts in both terminal regions, but total graft volume, assessed stereologically, showed a significant negative correlation with error scores. Rats with behaviourally effective grafts, like controls, were disrupted in the place task when tested in dim light which obscured extra-maze spatial cues. Lesioned rats were not affected by change in lighting. Grafts of cholinergic-poor fetal hippocampal tissue did not improve radial maze performance; neither did grafts of cholinergic-rich tissue placed within the host basal forebrain lesion sites. In rats with cholinergic-rich terminal grafts, cortical and hippocampal choline acetyltransferase activity was restored to control level, commensurate with site of transplant, whereas it was significantly reduced in lesioned animals and those with functionally ineffective grafts. The indiscriminate error pattern and insensitivity to changes in lighting shown by lesioned rats suggested that lesioning primarily disrupted attention rather than short- or long-term spatial or associative memory processes. Since rats with cholinergic-rich grafts showed both reduced errors and recovery of stimulus control, the data indicated that grafts affected information processing, rather than changes in motor or motivational processes. Changes in choline acetyltransferase activity and the behavioural efficacy of cholinergic-rich grafts are consistent with the involvement of acetylcholine in the behavioural deficits and recovery displayed by lesioned and grafted groups, but do not rule out contributions from other factors. The equipotency of grafts within each terminal region suggests also that there may be a considerable degree of functional cooperation between the two branches of the forebrain cholinergic projection system. Functional recovery may involve local, nonspecific synaptic or paracrine mechanisms within the target regions, since grafts were efficacious only when placed in the terminal areas, but not when sited homotopically in the basal forebrain, indicating that they did not achieve any functionally significant structural repair to the host brain at that site.     

A model for primitive neuroectodermal tumors in transgenic neural transplants harboring the SV40 large T antigen.

Using retrovirus-mediated transfer of the SV40 virus large T antigen into neural transplants, we have observed a high incidence of primitive neuroectodermal tumors (PNET). These neoplasms developed in 8 of 14 (57%) neural grafts after latency periods of 176 to 311 days. Histopathologically, the tumors exhibited features of human PNET such as formation of neuroblastic rosettes and immunocytochemical evidence for neuronal differentiation, synaptogenesis, and focal astrocytic differentiation. All neoplasms showed a striking migratory potential. The presence of the large T gene in the tumors was demonstrated by polymerase chain reaction-mediated amplification of a specific 242 bp segment of large T and DNA sequence analysis. Large T antigen was identified in tissue sections using an immunocytochemical reaction with the monoclonal antibody Pab 108. Cell lines were established from several tumors and subjected to G418 selection. Secondary tumors induced by intracerebral transplantation of these cells retained the characteristic morphological and immunocytochemical properties of PNETs. These experiments demonstrate a considerable transforming potential of SV40 large T antigen for neural precursor cells. The long latency period suggests that neoplastic transformation initiated by the large T gene requires additional spontaneous mutations of cooperating cellular genes. Because the mechanism of transformation by large T antigen appears to involve complex formation with and inactivation of cellular tumor suppressor gene products, these cell lines may serve as an interesting tool to search for novel neural tumor suppressor genes.     

Morphological aspects of the vascularization in intraventricular neural transplants from embryo to embryo

Intraventricular transplants of neural tissues were performed in ovo from embryo to embryo. Fragments of the nervous wall of the optic lobe (tectum) from 14-day chick or 12-day quail embryos (donor) were inserted into the ventricle of the right optic lobe of 6-day chick or 5-day quail embryos (host). Chick-to-chick, chick-to-quail and quail-to-chick grafts were carried out. The vascularization changes occurring in the host tectum and in the grafted neural tissues were analysed under light, transmission, and scanning electron microscopes and by morphometric methods. In the host embryo tectum, the neural graft stimulates a statistically significant increment in vessel density and a vessel sprouting into the ventricle of the optic lobe. The vascular sprouts reach the transplanted tissue and establish connections with its native microvasculature. The chick-to-quail and quail-to chick grafts, submitted to immunoreaction with a quailspecific antibody which recognizes an antigen (MB1) present on endothelial cells, indicate that re-establishment of the circulation in the graft depends upon anastomoses between host and donor vasculatures and the rapid new growth of host-derived and donor-native vessels. The presence of macrophage-like cells escorting the new-growing vessels suggests that these cells are involved in the host and donor tissue angiogenesis.     

Robust neural integration from retinal transplants in mice deficient in GFAP and vimentin

With recent progress in neuroscience and stem-cell research, neural transplantation has emerged as a promising therapy for treating CNS diseases. The success of transplantation has been limited, however, by the restricted ability of neural implants to survive and establish neuronal connections with the host. Little is known about the mechanisms responsible for this failure. Neural implantation triggers reactive gliosis, a process accompanied by upregulation of intermediate filaments in astrocytes and formation of astroglial scar tissue. Here we show that the retinas of adult mice deficient in glial fibrillary acidic protein and vimentin, and consequently lacking intermediate filaments in reactive astrocytes and Müller cells, provide a permissive environment for grafted neurons to migrate and extend neurites. The transplanted cells integrated robustly into the host retina with distinct neuronal identity and appropriate neuronal projections. Our results indicate an essential role for reactive astroglial cells in preventing neural graft integration after transplantation.     

Membrane specializations and extracellular material associated with host astrocytes in peripheral neural transplants

The work of Aguayo and colleagues [Aguayo, David and Bray (1981) J. exp. Biol.95, 231–240] demonstrates that grafts of peripheral neural tissue are able to induce regenerative elongation of cut axons in the adult central nervous system. Elucidation of the mechanism of this response requires an understanding of the cellular interactions induced by these types of transplant. In previous studies [Zhou, Lawrence, Morris and Raisman (1986) Neuroscience17, 815–827; Zhou, Lindsay, Lawrence and Raisman (1986) Neuroscience17, 803–813] we have transplanted decapsulated adult superior cervical sympathetic ganglia or nodose ganglia into either the septal nuclei or the choroid fissure of adult syngeneic rat hosts. We found that host astrocytes invade the transplants along Schwann cell fascicles and around blood vessels. This raises the questions of what form the migrating astrocytes take, what routes they follow, and what is their fate.

In the present study we have taken advantage of the fact that at longer survivals astrocytes accumulate as “paravascular cuffs”, and we show that they have several specialized ultrastructural features, such as plasmalemmal caveolae, desmosomes, hemidesmosomes and accumulations of extracellular material. The specific stimuli inducing (or enhancing) these astrocytic specializations and their significance in relation to the wider morphogenetic events induced by peripheral neural transplants remain to be elucidated. However, the observations are further evidence of the remarkable mobility and plasticity of central astrocytes in transplantation situations, and in particular emphasize the involvement of the cell surface and its relationship to extracellular matrix.     

Receptor-mediated interleukin-2 gene transfer into human hepatoma cells.

Receptor-mediated gene delivery is an attractive method for gene transfer in vitro and shows promise for in vivo gene therapy applications. In the current study, we have selected the cytokine interleukin-2 (IL-2) gene to explore the feasibility of receptor-mediated gene transfer into human hepatocellular carcinoma HepG2 cells, using Epstein-Barr virus (EBV)-based vectors. We have developed a targeted DNA delivery system for the treatment of liver cancer by gene therapy. This system utilizes the hepatocyte-specific asialoglycoprotein receptor, which is uniquely expressed on liver cell membranes but not present on other cell types. Galactosylated histone, a ligand to the asialoglycoprotein receptors, was synthesized, and a new EBV-based expression vector bearing the human IL-2 cDNA was constructed and conjugated to the ligand through ionic interactions. The ligand/IL-2 DNA complex was able to bind specifically to cell-surface receptors on the target cell and, when incubated with HepG2 cells, resulted in elevated levels of IL-2 gene expression. These results indicate that therapeutic genes like IL-2 in ligand/DNA complex can be transferred into hepatoma cells via the hepatocyte receptor. This study constitutes an encouraging first step in the assessment of receptor-mediated gene transfer as a technique for gene therapy in liver cancer.