Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34+ cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

The Friend virus susceptibility 2 (Fv2) locus encodes a dominant host factor that confers susceptibility to Friend virus-induced erythroleukaemia in mice. We mapped Fv2 to a 1.0-Mb interval that also contained the gene (Ron) encoding the stem cell kinase receptor (Stk). A truncated form of Stk (Sf-stk), which was the most abundant form of Stk in Fv2-sensitive (Fv2ss) erythroid cells, was not expressed in Fv2 resistant (Fv2rr) cells. Enforced expression of Sf-stk conferred susceptibility to Friend disease, whereas targeted disruption of Ron caused resistance. We conclude that the Fv2 locus encodes Ron, and that a naturally expressed, truncated form of Stk confers susceptibility to Friend virus-induced erythroleukaemia.

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of ␤-thalassemia and sickle cell anemia. A treatment strategy using autologous stem celltargeted gene transfer of a ␥-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral-and lentiviral-based ␥-globin vectors for expression in transduced erythroid cell lines. Compared with ␥-globin, oncoretroviral vectors containing either a ␤-spectrin or ␤-globin promoter and the ␣-globin HS40 element, a ␥-globin lentiviral vector utilizing the ␤-globin promoter and elements from the ␤-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human ␥-globin was observed with levels of vector-encoded ␥-globin mRNA ranging from 9% to 19% of total murine ␣-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of ␤-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m␣ 2 h␥ 2 molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. ␥-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a ␥-globin lentiviral vector for gene therapy of ␤-thalassemia. IntroductionThe hemoglobin disorders are highly prevalent, recessive genetic diseases in which coinheritance of 2 defective globin alleles results in severe hematologic disease. In patients with sickle cell anemia, the beta chain of hemoglobin S contains a substitution of valine for glutamic acid at position 6. 1 This substitution results in a change in surface charge that predisposes deoxygenated HbS to polymerize, causing red cells to assume rigid sickled shapes leading to vaso-occlusion, painful crisis, and organ damage. Defective synthesis of ␤-globin in patients with severe ␤-thalassemia due to a variety of mutational mechanisms leads to the accumulation of aggregates of unpaired, insoluble ␣-chains that cause ineffective erythropoiesis, accelerated red cell destruction, and severe anemia. 2 Although palliative therapies improve the quality and duration of life for many individuals, overall treatment for these disorders remains unsatisfactory. A few patients with sickle cell disease and a somewhat larger number with ␤-thalassemia have been cured with bone marrow (BM) transplantation from HLAmatched siblings, but such treatment is available for only a small minority of patients. 3,4 These considerations have made the development of gene therapy for hemoglobin disorders a highly desired goal.Effective gene therapy for hemoglobi...

IntroductionThe ability to transfer genes into repopulating hematopoietic stem cells ex vivo and to achieve regulated expression in specific lineages following hematopoietic reconstitution would create many therapeutic opportunities. 1 Although the initial use of murine oncoretroviral vectors to transfer genes into primitive murine hematopoietic cells was reported 20 years ago, 2 translation of this approach to clinical application has been slow and has required considerable effort. Despite progress being achieved in the murine system with correction of single gene defects in murine models of human immunodeficiencies [3][4][5][6] and chronic granulomatous disease, 7,8 the much lower efficiency of gene transfer into human stem cells 1 has hampered success. The necessity for high-level oncoretroviral vector gene transfer to achieve therapeutic benefit, however, was circumvented in 2 recent clinical trials designed to cure severe combined immunodeficiency due to a deficiency of the common ␥-chain of the lymphoid cytokine receptor 9 or adenosine deaminase. 10 In these trials, a potent selective repopulating advantage of the gene-corrected lymphoid cells resulted in therapeutically relevant numbers of functional lymphocytes.Despite this success, 2 barriers appear to limit the ability of murine oncoretroviral vectors to achieve adequate transduction of primitive hematopoietic stem cells for treatment of other disorders in which the gene-corrected cells do not have a selective advantage. Because the human homolog of the receptor for murine ecotropic vector particles does not interact with the ecotropic envelope protein, amphotropic particles have been used in both human studies and in large animal models. The amphotropic receptor, however, is expressed at low levels on human stem cells. 11 Alternative envelopes have been tested, such as those derived from the gibbon ape leukemia virus, 12 feline endogenous virus (RD114), 13 or feline leukemia virus type C, 14 the receptors for which are expressed at higher levels on primitive hematopoietic cell populations. However, large animal studies have failed to clearly identify a superior pseudotype that consistently yields high-level stem cell gene transfer. These data suggest that a second barrier-namely, the requirement for mitosis to allow integration of the oncoretroviral vector genome 15 -along with the relative instability of the preintegration complex 16 may be the main limitations of oncoretroviral-mediated stem cell gene transfer. In an effort to induce stem cell cycling, cytokines such as stem cell factor (SCF), Flt-3 ligand (Flt3-L), and megakaryocyte growth and development factor (MGDF) 17,18 are added to culture medium and a fragment of fibronectin is used to colocalize vector particles and target cells, 19 leading to improved stem cell transduction efficiency in large animal models. 12,20,21 Nonetheless, there remains significant variability among animals, with many animals having low marking and only occasional animals having proportions of genetically modifi...