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.

Long-term expression of coagulation factor IX (FIX) has been observed in murine and canine models following administration of recombinant adeno-associated viral (rAAV) vectors into either the portal vein or muscle. These studies were designed to evaluate factors that influence rAAV-mediated FIX expression.

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...

The feasibility, safety, and efficacy of liverdirected gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 ؋ 10 12 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecularweight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B. IntroductionRecombinant adeno-associated viral vectors (rAAVs) hold great promise for the treatment of hemophilia B. Preclinical studies in murine and canine models of hemophilia B have demonstrated persistent therapeutic expression of factor IX (FIX) leading to correction of the bleeding phenotype after intramuscular or portal vein administration of rAAV. 1-7 Liver-targeted delivery of rAAV resulted in significantly higher levels of FIX than observed after intramuscular administration of an equivalent dose of vector in immunodeficient mice. 8 More important, the development of neutralizing antibodies (NAbs) to human FIX (hFIX) is more common after intramuscular injection of rAAV than after intraportal administration of the same dose of vector. [8][9][10][11] Mice with persistent hFIX expression after liver-targeted delivery of rAAV could not mount a substantial antibody (Ab) response when challenged repeatedly with hFIX antigen in Freund adjuvant, a result suggesting that the liver has a unique ability to induce immune tolerance to neoantigens. 12 Endothelial cells of the liver, which can efficiently process and cross-present exogenous soluble antigens to CD8 ϩ T cells, are intimately involved in antigenspecific immune tolerance after liver-targeted gene transfer with rAAV vectors. 13 The findings of sustained expression of hFIX at therapeutic levels after a single bolus injection of rAAV vector into the portal vein of mice [3][4][5][6]8 and the absence of toxicity aft...

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...

Substantial effort has been invested in developing methodologies for efficient gene transfer into human, repopulating, hematopoietic stem cells. Oncoretroviral vectors are limited by the lack of nuclear mitosis in quiescent stem cells during ex vivo transduction, whereas the preintegration complex of lentiviral vectors contains nuclear-localizing signals that permit genome integration without mitosis. We have developed a flexible and versatile system for generating lentiviral vector particles and have pseudotyped such particles with amphotropic, ecotropic, feline endogenous virus (RD114) or vesicular stomatitis virus (VSV-G) envelope proteins. Particles of all four types could be concentrated approximately 100-fold by ultracentrifugation or ultrafiltration. RD114 or amphotropic particles were more efficient than VSV-G-pseudotyped particles at transducing human cord blood CD34(+) cells and clonogenic progenitors within that population. Amphotropic particles transduced cytokine-mobilized, human peripheral blood CD34(+) cells capable of establishing hematopoiesis in immunodeficient mice more efficiently than the other two types of particles. We conclude that the use of amphotropic pseudotyped lentiviral vector particles rather than the commonly used VSV-G-pseudotyped particles should be considered in potential applications of lentiviral vectors for gene transfer into this therapeutically relevant target cell population.

Since increased fetal hemoglobin diminishes the severity of ␤-thalassemia and sickle cell anemia, a strategy using autologous, stem cell-targeted gene transfer of a ␥-globin gene may be therapeutically useful. We previously found that a ␥-globin lentiviral vector utilizing the ␤-globin promoter and elements from the ␤-globin locus control region (LCR) totaling 1.7 kb could correct murine ␤-thalassemia. However, therapeutic consistency was compromised by chromosomal position effects on vector expression. In contrast, we show here that the majority of animals that received transplants of ␤-thalassemic stem cells transduced with a new vector containing 3.2 kb of LCR sequences expressed high levels of fetal hemoglobin (17%-33%), with an average vector copy number of 1.3. This led to a mean 26 g/L (2.6 g/dL) increase in hemoglobin concentration and enhanced amelioration of other hematologic parameters. Analysis of clonal erythroid cells of secondary spleen colonies from mice that underwent transplantation demonstrated an increased resistance of the larger LCR vector to stable and variegating position effects. This trend was also observed for vector insertion sites located inside genes, where vector expression was often compromised, in contrast to intergenic sites, where higher levels of expression were observed. These data emphasize the importance of overcoming detrimental position effects for consistent therapeutic globin vector expression.

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